Zinc-binding Motif Research Articles

Identification of Polymerase and Proofreading Exonuclease Domains in the DNA Polymerases IA, IB and Nuclear-Encoded RNA Polymerase of the Plant Chloroplasts

Chloroplast plays a crucial role in all photosynthetic plants and converts the light energy to chemical energy. It is a semi-autonomous organelle and is mostly controlled by its own genome and partly by the nuclear imports. To replicate its own genome, it uses two DNA polymerases, viz. polymerases IA and IB. DNA polymerase IA showed 72.45% identity to polymerase IB, but only 35.35% identity to the E. coli DNA polymerase I. Multiple sequence alignment (MSA) analysis have shown that the DNA polymerases IA and IB and the E. coli DNA polymerase I possess almost identical active sites for polymerization and proofreading (PR) functions, suggesting their possible common evolutionary origin. The nuclear-encoded RNA polymerase (NEP) is imported from the nucleus and involves in the transcription of all the four subunits of the chloroplast RNA polymerase. The polymerase catalytic core of the DNA polymerases IA, IB and the NEP are remarkably conserved and is in close agreement with other DNA/RNA polymerases reported already, and possess a typical template-binding pair (-YG-), a basic catalytic amino acid (K) to initiate catalysis and a basic nucleotide selection amino acid R at -4 from K. The DNA polymerases IA and IB are very similar to prokaryotic DNA polymerases, except in possessing a zinc-binding motif (ZBM) in them, like the eukaryotic replicases. Interestingly, the PR exonucleases of all three polymerases belong to the DEDD-superfamily of exonucleases. The DNA polymerases IA and IB belong to the DEDD(Y)-subfamily, whereas the NEP belongs to the DEDD(H)-subfamily.

Identification and Functional Characterization of WRKY, PHD and MYB Three Salt Stress Responsive Gene Families in Mungbean (Vigna radiata L.).

WRKY-, PHD-, and MYB-like proteins are three important types of transcription factors in mungbeans, and play an important role in development and stress resistance. The genes' structures and characteristics were clearly reported and were shown to contain the conservative WRKYGQK heptapeptide sequence, Cys4-His-cys3 zinc binding motif, and HTH (helix) tryptophan cluster W structure, respectively. Knowledge on the response of these genes to salt stress is largely unknown. To address this issue, 83 VrWRKYs, 47 VrPHDs, and 149 VrMYBs were identified by using comparative genomics, transcriptomics, and molecular biology methods in mungbeans. An intraspecific synteny analysis revealed that the three gene families had strong co-linearity and an interspecies synteny analysis showed that mungbean and Arabidopsis were relatively close in genetic relationship. Moreover, 20, 10, and 20 genes showed significantly different expression levels after 15 days of salt treatment (p < 0.05; Log2 FC > 0.5), respectively. Additionally, in the qRT-PCR analysis, VrPHD14 had varying degrees of response to NaCl and PEG treatments after 12 h. VrWRKY49 was upregulated by ABA treatment, especially in the beginning (within 24 h). VrMYB96 was significantly upregulated in the early stages of ABA, NaCl, and PEG stress treatments (during the first 4 h). VrWRKY38 was significantly upregulated by ABA and NaCl treatments, but downregulated by PEG treatment. We also constructed a gene network centered on the seven DEGs under NaCl treatment; the results showed that VrWRKY38 was in the center of the PPI network and most of the homologous Arabidopsis genes of the interacted genes were reported to have response to biological stress. Candidate genes identified in this study provide abundant gene resources for the study of salt tolerance in mungbeans.

Rationally designed donepezil-based hydroxamates modulate Sig-1R and HDAC isoforms to exert anti-glioblastoma effects

The pursuit of activating the HDAC inhibitory template towards additional mechanisms spurred us to design dual modulators (Sig-1R agonist - HDAC inhibitor) via utilization of the core structural unit of donepezil (an FDA-approved anti-Alzheimer's agent) as a surface recognition part. Literature precedents coupled with our experience rendered us with several insights that led to the inclusion of chemically diverse linkers and hydroxamic acid (zinc-binding motif) as the other components of HDAC inhibitory pharmacophore. With this envisionment and clarity, donepezil-based HDAC inhibitory adducts were furnished and exhaustively explored for their anti-GBM efficacy. Resultantly, a magnificently potent HDAC inhibitor 10 [IC50 (HDAC6) = 2.7 nM, IC50 (HDAC2) = 0.71 μM] was pinpointed that was endowed with the ability to: i) exert cell growth inhibitory effects against Human U87MG GBM cells ii) cause death in TMZ-resistant GBM cells iii) induce subG1 arrest in GBM cells iv) prolong the survival of TMZ-resistant U87MG inoculated orthotopic mice (in-vivo studies) v) induce GBM cell apoptosis via binding to Sig-1R. Collectively, the results led to the identification of compound 10 as a tractable anti-GBM agent that deserves detailed investigation for the accomplishment of its candidature as a GBM therapeutic.

Structural and Functional Characterization of a New Bacterial Dipeptidyl Peptidase III Involved in Fruiting Body Formation in Myxobacteria.

Dipeptidyl peptidase III (DPP III) is a zinc-dependent enzyme that specifically hydrolyzes dipeptides from the N-terminal of different-length peptides, and it is involved in a number of physiological processes. Here, DPP III with an atypical pentapeptide zinc binding motif (HELMH) was identified from Corallococcus sp. EGB. It was shown that the activity of recombined CoDPP III was optimal at 50 °C and pH 7.0 with high thermostability up to 60 °C. Unique to CoDPP III, the crystal structure of the ligand-free enzyme was determined as a dimeric and closed form. The relatively small inter-domain cleft creates a narrower entrance to the substrate binding site and the unfavorable binding of the bulky naphthalene ring. The ectopic expression of CoDPP III in M. xanthus DK1622 resulted in a 12 h head start in fruiting body development compared with the wild type. Additionally, the A-signal prepared from the starving DK1622-CoDPP III rescued the developmental defect of the asgA mutant, and the fruiting bodies were more numerous and closely packed. Our data suggested that CoDPP III played a role in the fruiting body development of myxobacteria through the accumulation of peptides and amino acids to act as the A-signal.

The zinc-binding motif in tankyrases is required for the structural integrity of the catalytic ADP-ribosyltransferase domain.

Tankyrases are ADP-ribosylating enzymes that regulate many physiological processes in the cell and are considered promising drug targets for cancer and fibrotic diseases. The catalytic ADP-ribosyltransferase domain of tankyrases contains a unique zinc-binding motif of unknown function. Recently, this motif was suggested to be involved in the catalytic activity of tankyrases. In this work, we set out to study the effect of the zinc-binding motif on the activity, stability and structure of human tankyrases. We generated mutants of human tankyrase (TNKS) 1 and TNKS2, abolishing the zinc-binding capabilities, and characterized the proteins biochemically and biophysically in vitro. We further generated a crystal structure of TNKS2, in which the zinc ion was oxidatively removed. Our work shows that the zinc-binding motif in tankyrases is a crucial structural element which is particularly important for the structural integrity of the acceptor site. While mutation of the motif rendered TNKS1 inactive, probably due to introduction of major structural defects, the TNKS2 mutant remained active and displayed an altered activity profile compared to the wild-type.

Carboxypeptidase E Independently Changes Microtubule Glutamylation, Dendritic Branching, and Neuronal Migration.

Neuronal migration and dendritogenesis are dependent on dynamic changes to the microtubule (MT) network. Among various factors that regulate MT dynamics and stability, post-translational modifications (PTMs) of MTs play a critical role in conferring specificity of regulatory protein binding to MTs. Thus, it is important to understand the regulation of PTMs during brain development as multiple developmental processes are dependent on MTs. In this study, we identified that carboxypeptidase E (CPE) changes tubulin polyglutamylation, a major PTM in the brain, and we examine the impact of CPE-mediated changes to polyglutamylation on cortical neuron migration and dendrite morphology. We show, for the first time, that overexpression of CPE increases the level of polyglutamylated α-tubulin while knockdown decreases the level of polyglutamylation. We also demonstrate that CPE-mediated changes to polyglutamylation are dependent on the CPE zinc-binding motif and that this motif is necessary for CPE action on p150Glued localization. However, overexpression of a CPE mutant that does not increase MT glutamylation mimics the effects of overexpression of wild type CPE on dendrite branching. Furthermore, although overexpression of wild type CPE does not alter cortical neuron migration, overexpression of the mutant may act in a dominant-negative manner as it decreases the number of neurons that reach the cortical plate (CP), as we previously reported for CPE knockdown. Overall, our data suggest that CPE changes MT glutamylation and redistribution of p150Glued and that this function of CPE is independent of its role in shaping dendrite development but plays a partial role in regulating cortical neuron migration.

Zinc-chelating postsynaptic density-95 N-terminus impairs its palmitoyl modification.

Chemical synaptic transmission represents the most sophisticated dynamic process and is highly regulated with optimized neurotransmitter balance. Imbalanced transmitters can lead to transmission impairments, for example, intracellular zinc accumulation is a hallmark of degenerating neurons. However, the underlying mechanisms remain elusive. Postsynaptic density protein-95 (PSD-95) is a primary postsynaptic membrane-associated protein and the major scaffolding component in the excitatory postsynaptic densities, which performs substantial functions in synaptic development and maturation. Its membrane association induced by palmitoylation contributes largely to its regulatory functions at postsynaptic sites. Unlike other structural domains in PSD-95, the N-terminal region (PSD-95NT) is flexible and interacts with various targets, which modulates its palmitoylation of two cysteines (C3/C5) and glutamate receptor distributions in postsynaptic densities. PSD-95NT contains a putative zinc-binding motif (C2H2) with undiscovered functions. This study is the first effort to investigate the interaction between Zn2+ and PSD-95NT. The NMR titration of 15 N-labeled PSD-95NT by ZnCl2 was performed and demonstrated Zn2+ binds to PSD-95NT with a binding affinity (Kd ) in the micromolar range. The zinc binding was confirmed by fluorescence and mutagenesis assays, indicating two cysteines and two histidines (H24, H28) are critical residues for the binding. These results suggested the concentration-dependent zinc binding is likely to influence PSD-95 palmitoylation since the binding site overlaps the palmitoylation sites, which was verified by the mimic PSD-95 palmitoyl modification and intact cell palmitoylation assays. This study reveals zinc as a novel modulator for PSD-95 postsynaptic membrane association by chelating its N-terminal region, indicative of its importance in postsynaptic signaling.

Molecular basis of human ATM kinase inhibition.

Human checkpoint kinase ataxia telangiectasia-mutated (ATM) plays a key role in initiation of the DNA damage response following DNA double-strand breaks. ATM inhibition is a promising approach in cancer therapy, but, so far, detailed insights into the binding modes of known ATM inhibitors have been hampered due to the lack of high-resolution ATM structures. Using cryo-EM, we have determined the structure of human ATM to an overall resolution sufficient to build a near-complete atomic model and identify two hitherto unknown zinc-binding motifs. We determined the structure of the kinase domain bound to ATPγS and to the ATM inhibitors KU-55933 and M4076 at 2.8 Å, 2.8 Å and 3.0 Å resolution, respectively. The mode of action and selectivity of the ATM inhibitors can be explained by structural comparison and provide a framework for structure-based drug design.

Biochemical characterization of LysVpKK5 endolysin from a marine vibriophage

Endolysins have been proposed as a potential antibacterial alternative for aquaculture, especially against Vibrio; the bacterial-agents that most frequently cause disease. Although multiple marine vibriophages have been characterized to date, research on vibriophage endolysins is recent. In this study, biochemical characterization of LysVpKK5 endolysin encoded by Vibrio parahaemolyticus-infecting VpKK5 phage was performed. In silico analysis revealed that LysVpKK5 possesses a conserved amidase_2 domain with a zinc-binding motif of high structural similarity to T7 lysozyme (RMSD = 0.107 Å). Contrary to expectations, the activity was inhibited with Zn2+ and was improved with other divalent cations, especially Ca2+. It showed optimal muralytic activity at pH 10, and curiously, no lytic activity at pH ≤ 7 was recorded. As for the thermal stability test, the optimal activity was recorded at 30 °C; the higher residual activity was recorded at 4 °C, and was lost at ≥ 50 °C. On the other hand, increasing NaCl concentrations reduced the activity gradually; the optimal activity was recorded at 50 mM NaCl. On the other hand, the enzymatic activity at 0.5 M NaCl was approx 30% and of approx 50% in seawater. LysVpKK5 endolysin exhibited a higher activity on V. parahaemolyticus ATCC-17802 strain, in comparison with AHPND + strains.

Structural basis for the coiled-coil architecture of human CtIP

The DNA repair factor CtIP has a critical function in double-strand break (DSB) repair by homologous recombination, promoting the assembly of the repair apparatus at DNA ends and participating in DNA-end resection. However, the molecular mechanisms of CtIP function in DSB repair remain unclear. Here, we present an atomic model for the three-dimensional architecture of human CtIP, derived from a multi-disciplinary approach that includes X-ray crystallography, small-angle X-ray scattering (SAXS) and diffracted X-ray tracking (DXT). Our data show that CtIP adopts an extended dimer-of-dimers structure, in agreement with a role in bridging distant sites on chromosomal DNA during the recombinational repair. The zinc-binding motif in the CtIP N-terminus alters dynamically the coiled-coil structure, with functional implications for the long-range interactions of CtIP with DNA. Our results provide a structural basis for the three-dimensional arrangement of chains in the CtIP tetramer, a key aspect of CtIP function in DNA DSB repair.

The FtsHi Enzymes of Arabidopsis thaliana: Pseudo-Proteases with an Important Function.

FtsH metalloproteases found in eubacteria, animals, and plants are well-known for their vital role in the maintenance and proteolysis of membrane proteins. Their location is restricted to organelles of endosymbiotic origin, the chloroplasts, and mitochondria. In the model organism Arabidopsis thaliana, there are 17 membrane-bound FtsH proteases containing an AAA+ (ATPase associated with various cellular activities) and a Zn2+ metalloprotease domain. However, in five of those, the zinc-binding motif HEXXH is either mutated (FtsHi1, 2, 4, 5) or completely missing (FtsHi3), rendering these enzymes presumably inactive in proteolysis. Still, homozygous null mutants of the pseudo-proteases FtsHi1, 2, 4, 5 are embryo-lethal. Homozygous ftshi3 or a weak point mutant in FTSHi1 are affected in overall plant growth and development. This review will focus on the findings concerning the FtsHi pseudo-proteases and their involvement in protein import, leading to consequences in embryogenesis, seed growth, chloroplast, and leaf development and oxidative stress management.

"Computational and Functional Characterization of a Hemorrhagic Metalloproteinase Purified from Cerastes cerastes Venom".

Structural and functional aspects of snake venoms metalloproteinases (SVMPs) have been extensively studied due to their role in envenomation. However, in the detection of certain coagulation disorders these biomolecules have been used and applied for the production of new thrombolytic drugs. CcMP-II, a SVMP-II metalloproteinase with a hemorrhagic activity, isolated from the venom of Cerastes cerastes, its sequence of 472 amino acids was identified. Predicted 3D structure showed an arrangement of CcMP-II into two distinct domains: i) a metalloproteinase domain where the zinc-binding motif is found (HXXGHNLGIDH) in addition to the sequence Cys-Ile-Met (CIM) at the Met-turn and ii) disintegrin-like domain containing RGD motif. CcMP-II inhibits platelet aggregation induced by collagen in a dose-dependent manner with an IC50 value estimated of 0.11nM. This proteinase inhibits also aggregation of platelet stimulated by collagen even if the metal chelating agents (EDTA and 1, 10-phenontroline) are present suggesting that anti-aggregating effect is not due to its metalloproteinase domain, but to its disintegrin-like domain. Capillary pathological modifications caused by CcMP-II following intramuscular injection have as well been examined in mice. The key morphological alterations of the capillary vessels were clearly apparent from the ultrastructural study. The CcMP-II can play a key function in the pathogenesis of disorders that occurs following envenomation of Cerastes cerastes. The three-dimensional model of CcMP-II was built to explain structure-function relationships in ADAM/ADAMTs, a family of proteins having significant therapeutic potential. In order to explain structure-function relationships in ADAM / ADAMT, a family of proteins with considerable therapeutic potential, the three-dimensional model of CcMP-II was constructed.

The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity.

The activity of the TRPM7 channel is negatively regulated by intracellular Mg2+. We previously reported that oxidative stress enhances the inhibition of TRPM7 by intracellular Mg2+. Here, we aimed to clarify the mechanism underlying TRPM7 inhibition by hydrogen peroxide (H2O2). Site-directed mutagenesis of full-length TRPM7 revealed that none of the cysteines other than C1809 and C1813 within the zinc-binding motif of the TRPM7 kinase domain were involved in the H2O2-induced TRPM7 inhibition. Mutation of C1809 or C1813 prevented expression of full-length TRPM7 on the plasma membrane. We therefore developed an assay to functionally reconstitute full-length TRPM7 by coexpressing the TRPM7 channel domain (M7cd) and the TRPM7 kinase domain (M7kd) as separate proteins in HEK293 cells. When M7cd was expressed alone, the current was inhibited by intracellular Mg2+ more strongly than that of full-length TRPM7 and was insensitive to oxidative stress. Coexpression of M7cd and M7kd attenuated the inhibition by intracellular Mg2+ and restored sensitivity to oxidative stress, indicating successful reconstitution of a full-length TRPM7-like current. We observed a similar effect when M7cd was coexpressed with the kinase-inactive mutant M7kd-K1645R, suggesting that the kinase activity is not essential for the reconstitution. However, coexpression of M7cd and M7kd carrying a mutation at either C1809 or C1813 failed to restore the full-length TRPM7-like current. No reconstitution was observed when using M7kd carrying a mutation at H1750 and H1807, which are involved in the zinc-binding motif formation with C1809 and C1813. These data suggest that the zinc-binding motif is essential for the intracellular Mg2+-dependent regulation of the TRPM7 channel activity by its kinase domain and that the cysteines in the zinc-binding motif play a role in the oxidative stress response of TRPM7.

Porcine TRIM21 RING-finger E3 ubiquitin ligase is essential for anti-PRRSV activity

Porcine reproductive and respiratory syndrome (PRRS) causes substantial economic losses to the global pig industry. Members of the tripartite motif (TRIM) family are the important effectors of the innate immune response against viral infections. We have previously characterized the entire porcine TRIM (pTRIM) family, and predicted pTRIM5, 14, 21, 25 and 38 as host restriction factors against PRRSV infection. However, little is known about whether and how pTRIMs restrict the infection of PRRSV. In this study, we firstly performed the amino acid alignments of the RING domain of pTRIM5, 21, 25 and 38, and found that pTRIM proteins contained the characteristic consensus C3HC4 type zinc-binding motif which is important for the ubiquitination function. Then we detected the mRNA changes of pTRIMs in porcine alveolar macrophages (PAMs) by transcriptome sequencing after PRRSV infection in piglets. Transcriptional profiles showed that the expression of pTRIM5, 21 and 26 was significantly (P < 0.05) up-regulated, consistent with their expression in vitro. Finally, as the most up-regulated gene after PRRSV infection both in vivo and in vitro, pTRIM21 was investigated for its anti-PRRSV activity in immortalized PAMs (iPAMs) in two aspects: knockdown and overexpression of pTRIM21. Knockdown of endogenic pTRIM21 could significantly promote PRRSV replication at 12 and 24 h post infection in iPAMs. Meanwhile, overexpression of pTRIM21 could significantly suppress PRRSV replication but not affect its attachment and endocytosis. Moreover, pTRIM21 RING-finger E3 ubiquitin ligase was essential for anti-PRRSV activity. Our data enhance our understanding of the pTRIMs against PRRSV infection, which may help us develop novel therapeutic tools to control PRRSV.

Midgut aminopeptidase N expression profile in castor semilooper (Achaea janata) during sublethal Cry toxin exposure

The midgut of lepidopteran larvae is a multifunctional tissue that performs roles in digestion, absorption, immunity, transmission of pathogens and interaction with ingested various molecules. The proteins localized at the inner apical brush border membrane are primarily digestive proteases, but some of them, like aminopeptidase N, alkaline phosphatase, cadherins, ABC transporter C2, etc., interact with Crystal (Cry) toxins produced by Bacillus thuringiensis (Bt). In the present study, aminopeptidase N (APN) was characterized as Cry-toxin-interacting protein in the larval midgut of castor semilooper, Achaea janata. Transcriptomic and proteomic analyses revealed the presence of multiple isoforms of APNs (APN1, 2, 4, 6 and 9) which have less than 40% sequence similarity but show the presence of characteristic 'GAMENEG' and zinc-binding motifs. Feeding a sublethal dose of Cry toxin caused differential expression of various APN isoform. Further, 6thgeneration Cry-toxin-exposed larvae showed reduced expression of APN2. This report suggests that A. janata larvae exploit altered expression of APNs to overcome the deleterious effects of Cry toxicity, which might facilitate toxin tolerance in the long run.

Pragmatic recruitment of memantine as the capping group for the design of HDAC inhibitors: A preliminary attempt to unravel the enigma of glioblastoma.

Hurdled and marred by the notorious nature of glioblastomas (GBM) in terms of resistance to therapy and limited drug delivery into the brain, the anti-GBM drug pipeline is required to be loaded with mechanistically diverse agents. The consideration of HDAC inhibition as a prudent approach to circumvent the resistance issue in GBM spurred us to pragmatically design and synthesizes hydroxamic acids endowed with CNS penetrating ability. By virtue of the blood brain barrier permeability (BBB), memantine was envisioned as an appropriate CAP component for the construction of the HDAC inhibitors. Diverse linkers were stapled for the tetheration of the zinc binding motif with the CAP group to pinpoint an appropriate combination (CAP and linker) that could confer inhibitory preference to HDAC6 isoform (overexpressed in GBM). Resultantly, hydroxamic acid 16 was identified as a promising compound that elicited striking antiproliferative effects against Human U87MG GBM cells as well as TMZ-resistant GBM cells and P1S cells, a concurrent chemo radiotherapy (CCRT)-resistant/patient-derived glioma cell line mediated through preferential HDAC6 inhibition (IC50=5.42nM). Furthermore, 16 exerted cell cycle arrest at G2 phase, induced apoptosis in GBM cells at high concentration and exhibited high BBB permeability. To add on, in-vivo study revealed that the administration of compound 16 prolonged the survival of TMZ-resistant U87MG inoculated orthotopic mice. Overall, the cumulative results indicate that 16 is a tractable CNS penetrant preferential HDAC6 inhibitor that might emerge as a potent weapon against GBM.

Evolution of Ribosomal Protein S14 Demonstrated by the Reconstruction of Chimeric Ribosomes in Bacillus subtilis.

Ribosomal protein S14 can be classified into three types. The first, the C+ type has a Zn2+ binding motif and is ancestral. The second and third are the C- short and C- long types, neither of which contain a Zn2+ binding motif and which are ca. 90 residues and 100 residues in length, respectively. In the present study, the C+ type S14 from Bacillus subtilis ribosomes (S14BsC+) were completely replaced by the heterologous C- long type of S14 from Escherichia coli (S14Ec) or Synechococcus elongatus (S14Se). Surprisingly, S14Ec and S14Se were incorporated fully into 70S ribosomes in B. subtilis However, the growth rates as well as the sporulation efficiency of the mutants harboring heterologous S14 were significantly decreased. In these mutants, the polysome fraction was decreased and the 30S and 50S subunits accumulated unusually, indicating that cellular translational activity of these mutants was decreased. In vitro analysis showed a reduction in the translational activity of the 70S ribosome fraction purified from these mutants. The abundance of ribosomal proteins S2 and S3 in the 30S fraction in these mutants was reduced while that of S14 was not significantly decreased. It seems likely that binding of heterologous S14 changes the structure of the 30S subunit, which causes a decrease in the assembly efficiency of S2 and S3, which are located near the binding site of S14. Moreover, we found that S3 from S. elongatus cannot function in B. subtilis unless S14Se is present.IMPORTANCE S14, an essential ribosomal protein, may have evolved to adapt bacteria to zinc-limited environments by replacement of a zinc-binding motif with a zinc-independent sequence. It was expected that the bacterial ribosome would be tolerant to replacement of S14 because of the previous prediction that the spread of C- type S14 involved horizontal gene transfer. In this study, we completely replaced the C+ type of S14 in B. subtilis ribosome with the heterologous C- long type of S14 and characterized the resulting chimeric ribosomes. Our results suggest that the B. subtilis ribosome is permissive for the replacement of S14, but coevolution of S3 might be required to utilize the C- long type of S14 more effectively.

Replacement of S14 Protein in Ribosomes of Zinc-Starved Mycobacteria Reduces Spectinamide Sensitivity.

Zinc is an essential micronutrient for mycobacteria, and its depletion induces multiple adaptive changes in cellular physiology, the most remarkable of which are remodeling and hibernation of ribosomes. Ribosome remodeling, induced upon relatively moderate depletion of zinc, involves replacement of multiple ribosomal proteins containing the zinc-binding CXXC motif (called C+ r proteins) by their motif-free C- paralogs. Severe zinc depletion induces binding of mycobacterial protein Y (Mpy) to the 70S C- ribosome, thereby stabilizing the ribosome in an inactive state that is also resistant to kanamycin and streptomycin. Because the Mpy binding region on the ribosome is proximal to the binding pocket of spectinamides (Spa), the preclinical drug candidates for tuberculosis, we addressed the impact of remodeling and hibernation of ribosomes on Spa sensitivity. We report here that while Mpy binding has no significant effect on Spa sensitivity to the ribosome, replacement of S14C+ with its C- counterpart reduces the binding affinity of the drug by ∼2-fold, causing increased Spa tolerance in Mycobacterium smegmatis and Mycobacterium tuberculosis cells harboring the C- ribosome. The altered interaction between Spa and ribosomes likely results from new contact points for D67 and R83 residues of S14C- with U1138 and C1184 of 16S rRNA helix 34, respectively. Given that M. tuberculosis induces ribosome remodeling during progression from the acute to chronic phase of lung infection, our findings highlight new considerations in the development of Spa as effective drugs against tuberculosis.

Maedi–visna virus Vif protein uses motifs distinct from HIV-1 Vif to bind zinc and the cofactor required for A3 degradation

The mammalian apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3 or A3) family of cytidine deaminases restrict viral infections by mutating viral DNA and impeding reverse transcription. To overcome this antiviral activity, most lentiviruses express a viral accessory protein called the virion infectivity factor (Vif), which recruits A3 proteins to cullin–RING E3 ubiquitin ligases such as cullin-5 (Cul5) for ubiquitylation and subsequent proteasomal degradation. Although Vif proteins from primate lentiviruses such as HIV-1 utilize the transcription factor core-binding factor subunit beta as a noncanonical cofactor to stabilize the complex, the maedi–visna virus (MVV) Vif hijacks cyclophilin A (CypA) instead. Because core-binding factor subunit beta and CypA are both highly conserved among mammals, the requirement for two different cellular cofactors suggests that these two A3-targeting Vif proteins have different biochemical and structural properties. To investigate this topic, we used a combination of in vitro biochemical assays and in vivo A3 degradation assays to study motifs required for the MVV Vif to bind zinc ion, Cul5, and the cofactor CypA. Our results demonstrate that although some common motifs between the HIV-1 Vif and MVV Vif are involved in recruiting Cul5, different determinants in the MVV Vif are required for cofactor binding and stabilization of the E3 ligase complex, such as the zinc-binding motif and N- and C-terminal regions of the protein. Results from this study advance our understanding of the mechanism of MVV Vif recruitment of cellular factors and the evolution of lentiviral Vif proteins.

Crystal structure and mutational analysis of the human TRIM7 B30.2 domain provide insights into the molecular basis of its binding to glycogenin-1

Tripartite motif (TRIM)7 is an E3 ubiquitin ligase that was first identified through its interaction with glycogenin-1 (GN1), the autoglucosyltransferase that initiates glycogen biosynthesis. A growing body of evidence indicates that TRIM7 plays an important role in cancer development, viral pathogenesis, and atherosclerosis and, thus, represents a potential therapeutic target. TRIM family proteins share a multidomain architecture with a conserved N-terminal TRIM and a variable C-terminal domain. Human TRIM7 contains the canonical TRIM motif and a B30.2 domain at the C terminus. To contribute to the understanding of the mechanism of action of TRIM7, we solved the X-ray crystal structure of its B30.2 domain (TRIM7B30.2) in two crystal forms at resolutions of 1.6 Å and 1.8 Å. TRIM7B30.2 exhibits the typical B30.2 domain fold, consisting of two antiparallel β-sheets of seven and six strands, arranged as a distorted β-sandwich. Furthermore, two long loops partially cover the concave face of the β-sandwich defined by the β-sheet of six strands, thus forming a positively charged cavity. We used sequence conservation and mutational analyses to provide evidence of a putative binding interface for GN1. These studies showed that Leu423, Ser499, and Cys501 of TRIM7B30.2 and the C-terminal 33 amino acids of GN1 are critical for this binding interaction. Molecular dynamics simulations also revealed that hydrogen bond and hydrophobic interactions play a major role in the stability of a modeled TRIM7B30.2-GN1 C-terminal peptide complex. These data provide useful information that could be used to target this interaction for the development of potential therapeutic agents.