ABSTRACT
In response to genotoxic stress, the tumor suppressor p53 acts as a transcription factor by regulating the expression of genes critical for cancer prevention. Mutations in the gene encoding p53 are associated with cancer development. PRIMA-1 and eprenetapopt (APR-246/PRIMA-1MET) are small molecules that are converted into the biologically active compound, methylene quinuclidinone (MQ), shown to reactivate mutant p53 by binding covalently to cysteine residues. Here, we investigate the structural basis of mutant p53 reactivation by MQ based on a series of high-resolution crystal structures of cancer-related and wild-type p53 core domains bound to MQ in their free state and in complexes with their DNA response elements. Our data demonstrate that MQ binds to several cysteine residues located at the surface of the core domain. The structures reveal a large diversity in MQ interaction modes that stabilize p53 and its complexes with DNA, leading to a common global effect that is pertinent to the restoration of non-functional p53 proteins.
Subject(s)
Antineoplastic Agents/pharmacology , Aza Compounds/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Neoplasms/drug therapy , Quinuclidines/pharmacology , Tumor Suppressor Protein p53/agonists , Antineoplastic Agents/therapeutic use , Aza Compounds/chemistry , Aza Compounds/therapeutic use , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Crystallography, X-Ray , Humans , Loss of Function Mutation/drug effects , Neoplasms/genetics , Protein Domains/drug effects , Quinuclidines/chemistry , Quinuclidines/therapeutic use , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/isolation & purification , Tumor Suppressor Protein p53/ultrastructureABSTRACT
Macrolides have been effective clinical antibiotics for over 70 years. They inhibit protein biosynthesis in bacterial pathogens by narrowing the nascent protein exit tunnel in the ribosome. The macrolide class of natural products consist of a macrolactone ring linked to one or more sugar molecules. Most of the macrolides used currently are semi-synthetic erythromycin derivatives, composed of a 14- or 15-membered macrolactone ring. Rapidly emerging resistance in bacterial pathogens is among the most urgent global health challenges, which render many antibiotics ineffective, including next-generation macrolides. To address this threat and advance a longer-term plan for developing new antibiotics, we demonstrate how 16-membered macrolides overcome erythromycin resistance in clinically isolated Staphylococcus aureus strains. By determining the structures of complexes of the large ribosomal subunit of Deinococcus radiodurans (D50S) with these 16-membered selected macrolides, and performing anti-microbial studies, we identified resistance mechanisms they may overcome. This new information provides important insights toward the rational design of therapeutics that are effective against drug resistant human pathogens.
Subject(s)
Macrolides/chemistry , Micromonospora/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Erythromycin/chemistry , Humans , Macrolides/pharmacology , Microbial Sensitivity Tests , Protein Synthesis Inhibitors/pharmacology , Ribosomes/drug effects , Staphylococcus aureus/drug effects , Staphylococcus aureus/pathogenicityABSTRACT
Allostery is a pervasive principle to regulate protein function. Growing evidence suggests that also DNA is capable of transmitting allosteric signals. Yet, whether and how DNA-mediated allostery plays a regulatory role in gene expression remained unclear. Here, we show that DNA indeed transmits allosteric signals over long distances to boost the binding cooperativity of transcription factors. Phenotype switching in Bacillus subtilis requires an all-or-none promoter binding of multiple ComK proteins. We use single-molecule FRET to demonstrate that ComK-binding at one promoter site increases affinity at a distant site. Cryo-EM structures of the complex between ComK and its promoter demonstrate that this coupling is due to mechanical forces that alter DNA curvature. Modifications of the spacer between sites tune cooperativity and show how to control allostery, which allows a fine-tuning of the dynamic properties of genetic circuits.
Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/genetics , DNA, Bacterial/chemistry , Gene Expression Regulation, Bacterial , Transcription Factors/genetics , Allosteric Regulation/genetics , Binding Sites/genetics , DNA, Bacterial/genetics , Gene Regulatory Networks , Nucleic Acid Conformation , Phenotype , Promoter Regions, Genetic/geneticsABSTRACT
Ribosomal RNA is the central component of the ribosome, mediating its functional and architectural properties. Here, we report the cryo-EM structure of a highly divergent cytoplasmic ribosome from the single-celled eukaryotic alga Euglena gracilis. The Euglena large ribosomal subunit is distinct in that it contains 14 discrete rRNA fragments that are assembled non-covalently into the canonical ribosome structure. The rRNA is substantially enriched in post-transcriptional modifications that are spread far beyond the catalytic RNA core, contributing to the stabilization of this highly fragmented ribosome species. A unique cluster of five adenosine base methylations is found in an expansion segment adjacent to the protein exit tunnel, such that it is positioned for interaction with the nascent peptide. As well as featuring distinctive rRNA expansion segments, the Euglena ribosome contains four novel ribosomal proteins, localized to the ribosome surface, three of which do not have orthologs in other eukaryotes.
Subject(s)
Euglena gracilis/chemistry , RNA, Ribosomal/chemistry , Ribosomes/chemistry , Cryoelectron Microscopy , Cytoplasm/chemistry , Euglena gracilis/genetics , Euglena gracilis/metabolism , Models, Molecular , RNA Processing, Post-Transcriptional , RNA, Ribosomal/metabolism , Ribosomal Proteins/chemistryABSTRACT
The tumor suppressor p53 acts as a transcription factor recognizing diverse DNA response elements (REs). Previous structural studies of p53-DNA complexes revealed non-canonical Hoogsteen geometry of A/T base pairs at conserved CATG motifs leading to changes in DNA shape and its interface with p53. To study the effects of DNA shape on binding characteristics, we designed REs with modified base pairs "locked" into either Hoogsteen or Watson-Crick form. Here we present crystal structures of these complexes and their thermodynamic and kinetic parameters, demonstrating that complexes with Hoogsteen base pairs are stabilized relative to those with all-Watson-Crick base pairs. CATG motifs are abundant in p53REs such as GADD45 and p53R2 related to cell-cycle arrest and DNA repair. The high-resolution structures of these complexes validate their propensity to adopt the unique Hoogsteen-induced structure, thus providing insights into the functional role of DNA shape and broadening the mechanisms that contribute to DNA recognition by proteins.
Subject(s)
DNA/chemistry , DNA/metabolism , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/metabolism , Binding Sites , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Humans , Hydrogen Bonding , Kinetics , Models, Molecular , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Nucleic Acid Conformation , Protein Binding , Protein Conformation , Protein Stability , Response Elements , Ribonucleotide Reductases/chemistry , Ribonucleotide Reductases/metabolismABSTRACT
Leishmania is a single-celled eukaryotic parasite afflicting millions of humans worldwide, with current therapies limited to a poor selection of drugs that mostly target elements in the parasite's cell envelope. Here we determined the atomic resolution electron cryo-microscopy (cryo-EM) structure of the Leishmania ribosome in complex with paromomycin (PAR), a highly potent compound recently approved for treatment of the fatal visceral leishmaniasis (VL). The structure reveals the mechanism by which the drug induces its deleterious effects on the parasite. We further show that PAR interferes with several aspects of cytosolic translation, thus highlighting the cytosolic rather than the mitochondrial ribosome as the primary drug target. The results also highlight unique as well as conserved elements in the PAR-binding pocket that can serve as hotspots for the development of novel therapeutics.
Subject(s)
Leishmania/metabolism , Paromomycin/metabolism , Ribosomes/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites/genetics , Cryoelectron Microscopy , Cytosol/drug effects , Cytosol/metabolism , Humans , Leishmania/genetics , Leishmania/ultrastructure , Models, Molecular , Paromomycin/chemistry , Paromomycin/pharmacology , Protein Biosynthesis/drug effects , RNA, Ribosomal/chemistry , RNA, Ribosomal/metabolism , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolism , Ribosomes/chemistry , Ribosomes/ultrastructure , Sequence Homology, Amino AcidABSTRACT
Antimicrobial resistance within a wide range of pathogenic bacteria is an increasingly serious threat to global public health. Among these pathogenic bacteria are the highly resistant, versatile and possibly aggressive bacteria, Staphylococcus aureus. Lincosamide antibiotics were proved to be effective against this pathogen. This small, albeit important group of antibiotics is mostly active against Gram-positive bacteria, but also used against selected Gram-negative anaerobes and protozoa. S. aureus resistance to lincosamides can be acquired by modifications and/or mutations in the rRNA and rProteins. Here, we present the crystal structures of the large ribosomal subunit of S. aureus in complex with the lincosamides lincomycin and RB02, a novel semisynthetic derivative and discuss the biochemical aspects of the in vitro potency of various lincosamides. These results allow better understanding of the drugs selectivity as well as the importance of the various chemical moieties of the drug for binding and inhibition.
Subject(s)
Lincosamides/pharmacology , Ribosome Subunits, Large, Bacterial/drug effects , Staphylococcus aureus/drug effects , Benzamides/chemistry , Benzamides/pharmacology , Binding Sites , Clindamycin/chemistry , Clindamycin/pharmacology , Crystallization , Crystallography, X-Ray , Drug Resistance, Microbial , Galactosides/chemistry , Galactosides/pharmacology , Hydrogen Bonding , Lincomycin/chemistry , Lincomycin/pharmacology , Lincosamides/chemistry , Molecular Structure , Ribosome Subunits, Large, Bacterial/ultrastructure , Staphylococcus aureus/ultrastructure , Static Electricity , Structure-Activity RelationshipABSTRACT
Erythromycin is a clinically useful antibiotic that binds to an rRNA pocket in the ribosomal exit tunnel. Commonly, resistance to erythromycin is acquired by alterations of rRNA nucleotides that interact with the drug. Mutations in the ß hairpin of ribosomal protein uL22, which is rather distal to the erythromycin binding site, also generate resistance to the antibiotic. We have determined the crystal structure of the large ribosomal subunit from Deinococcus radiodurans with a three amino acid insertion within the ß hairpin of uL22 that renders resistance to erythromycin. The structure reveals a shift of the ß hairpin of the mutated uL22 toward the interior of the exit tunnel, triggering a cascade of structural alterations of rRNA nucleotides that propagate to the erythromycin binding pocket. Our findings support recent studies showing that the interactions between uL22 and specific sequences within nascent chains trigger conformational rearrangements in the exit tunnel.
Subject(s)
Bacterial Proteins/chemistry , Ribosomal Proteins/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Deinococcus/chemistry , Erythromycin/chemistry , Erythromycin/pharmacology , Mutation , Protein Binding , RNA, Ribosomal/chemistry , RNA, Ribosomal/metabolism , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolismABSTRACT
The increasing appearance of pathogenic bacteria with antibiotic resistance is a global threat. Consequently, clinically available potent antibiotics that are active against multidrug resistant pathogens are becoming exceedingly scarce. Ribosomes are a main target for antibiotics, and hence are an objective for novel drug development. Lefamulin, a semi-synthetic pleuromutilin compound highly active against multi-resistant pathogens, is a promising antibiotic currently in phase III trials for the treatment of community-acquired bacterial pneumonia in adults. The crystal structure of the Staphylococcus aureus large ribosomal subunit in complex with lefamulin reveals its protein synthesis inhibition mechanism and the rationale for its potency. In addition, analysis of the bacterial and eukaryotes ribosome structures around the pleuromutilin binding pocket has elucidated the key for the drug's selectivity.
Subject(s)
Anti-Bacterial Agents , Ribosome Subunits, Large, Bacterial , Staphylococcus aureus , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/biosynthesis , Clinical Trials, Phase III as Topic , Diterpenes/chemistry , Diterpenes/pharmacology , Humans , Polycyclic Compounds , Protein Biosynthesis/drug effects , Ribosome Subunits, Large, Bacterial/chemistry , Ribosome Subunits, Large, Bacterial/metabolism , Staphylococcal Infections/drug therapy , Staphylococcal Infections/metabolism , Staphylococcal Infections/pathology , Staphylococcus aureus/chemistry , Staphylococcus aureus/growth & development , PleuromutilinsABSTRACT
Two structurally unique ribosomal antibiotics belonging to the orthosomycin family, avilamycin and evernimicin, possess activity against Enterococci, Staphylococci, and Streptococci, and other Gram-positive bacteria. Here, we describe the high-resolution crystal structures of the eubacterial large ribosomal subunit in complex with them. Their extended binding sites span the A-tRNA entrance corridor, thus inhibiting protein biosynthesis by blocking the binding site of the A-tRNA elbow, a mechanism not shared with other known antibiotics. Along with using the ribosomal components that bind and discriminate the A-tRNA-namely, ribosomal RNA (rRNA) helices H89, H91, and ribosomal proteins (rProtein) uL16-these structures revealed novel interactions with domain 2 of the CTC protein, a feature typical to various Gram-positive bacteria. Furthermore, analysis of these structures explained how single nucleotide mutations and methylations in helices H89 and H91 confer resistance to orthosomycins and revealed the sequence variations in 23S rRNA nucleotides alongside the difference in the lengths of the eukaryotic and prokaryotic α1 helix of protein uL16 that play a key role in the selectivity of those drugs. The accurate interpretation of the crystal structures that could be performed beyond that recently reported in cryo-EM models provide structural insights that may be useful for the design of novel pathogen-specific antibiotics, and for improving the potency of orthosomycins. Because both drugs are extensively metabolized in vivo, their environmental toxicity is very low, thus placing them at the frontline of drugs with reduced ecological hazards.
Subject(s)
Aminoglycosides/pharmacology , Bacterial Proteins/drug effects , Binding Sites/drug effects , Oligosaccharides/pharmacology , RNA, Transfer/drug effects , Ribosomal Proteins/drug effects , Aminoglycosides/chemistry , Anti-Bacterial Agents/pharmacology , Crystallography, X-Ray , Drug Resistance, Bacterial/drug effects , Drug Resistance, Bacterial/genetics , Gram-Positive Bacteria/drug effects , Microbial Sensitivity Tests , Models, Molecular , Mutation , Nucleic Acid Conformation , Oligosaccharides/chemistry , Protein Biosynthesis/drug effects , RNA, Ribosomal , RNA, Ribosomal, 23S/drug effects , RNA, Ribosomal, 23S/genetics , RNA, Transfer/metabolism , Ribosomal Proteins/metabolism , Ribosomes/drug effects , Ribosomes/metabolism , Sequence Alignment , Species SpecificityABSTRACT
Most ribosomal antibiotics obstruct distinct ribosomal functions. In selected cases, in addition to paralyzing vital ribosomal tasks, some ribosomal antibiotics are involved in cellular regulation. Owing to the global rapid increase in the appearance of multi-drug resistance in pathogenic bacterial strains, and to the extremely slow progress in developing new antibiotics worldwide, it seems that, in addition to the traditional attempts at improving current antibiotics and the intensive screening for additional natural compounds, this field should undergo substantial conceptual revision. Here, we highlight several contemporary issues, including challenging the common preference of broad-range antibiotics; the marginal attention to alterations in the microbiome population resulting from antibiotics usage, and the insufficient awareness of ecological and environmental aspects of antibiotics usage. We also highlight recent advances in the identification of species-specific structural motifs that may be exploited for the design and the creation of novel, environmental friendly, degradable, antibiotic types, with a better distinction between pathogens and useful bacterial species in the microbiome. Thus, these studies are leading towards the design of "pathogen-specific antibiotics," in contrast to the current preference of broad range antibiotics, partially because it requires significant efforts in speeding up the discovery of the unique species motifs as well as the clinical pathogen identification.
ABSTRACT
Leishmania is a single-cell eukaryotic parasite of the Trypanosomatidae family, whose members cause an array of tropical diseases. The often fatal outcome of infections, lack of effective vaccines, limited selection of therapeutic drugs, and emerging resistant strains, underline the need to develop strategies to combat these pathogens. The Trypanosomatid ribosome has recently been highlighted as a promising therapeutic target due to structural features that are distinct from other eukaryotes. Here, we present the 2.8-Å resolution structure of the Leishmania donovani large ribosomal subunit (LSU) derived from a cryo-EM map, further enabling the structural observation of eukaryotic rRNA modifications that play a significant role in ribosome assembly and function. The structure illustrates the unique fragmented nature of leishmanial LSU rRNA and highlights the irregular distribution of rRNA modifications in Leishmania, a characteristic with implications for anti-parasitic drug development.
Subject(s)
Leishmania donovani , Ribosome Subunits, Large/chemistry , Cryoelectron Microscopy , Models, Molecular , Nucleic Acid Conformation , Protein Structure, Quaternary , Protozoan Proteins/chemistry , RNA, Protozoan/chemistry , RNA, Ribosomal/chemistry , Ribosomal Proteins/chemistry , Ribosome Subunits, Large/ultrastructureABSTRACT
The emergence of bacterial multidrug resistance to antibiotics threatens to cause regression to the preantibiotic era. Here we present the crystal structure of the large ribosomal subunit from Staphylococcus aureus, a versatile Gram-positive aggressive pathogen, and its complexes with the known antibiotics linezolid and telithromycin, as well as with a new, highly potent pleuromutilin derivative, BC-3205. These crystal structures shed light on specific structural motifs of the S. aureus ribosome and the binding modes of the aforementioned antibiotics. Moreover, by analyzing the ribosome structure and comparing it with those of nonpathogenic bacterial models, we identified some unique internal and peripheral structural motifs that may be potential candidates for improving known antibiotics and for use in the design of selective antibiotic drugs against S. aureus.
Subject(s)
Ribosomes/metabolism , Staphylococcus aureus/metabolism , Protein Conformation , RNA, Ribosomal/metabolism , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolismABSTRACT
Leishmaniasis comprises an array of diseases caused by pathogenic species of Leishmania, resulting in a spectrum of mild to life-threatening pathologies. Currently available therapies for leishmaniasis include a limited selection of drugs. This coupled with the rather fast emergence of parasite resistance, presents a dire public health concern. Paromomycin (PAR), a broad-spectrum aminoglycoside antibiotic, has been shown in recent years to be highly efficient in treating visceral leishmaniasis (VL)-the life-threatening form of the disease. While much focus has been given to exploration of PAR activities in bacteria, its mechanism of action in Leishmania has received relatively little scrutiny and has yet to be fully deciphered. In the present study we present an X-ray structure of PAR bound to rRNA model mimicking its leishmanial binding target, the ribosomal A-site. We also evaluate PAR inhibitory actions on leishmanial growth and ribosome function, as well as effects on auditory sensory cells, by comparing several structurally related natural and synthetic aminoglycoside derivatives. The results provide insights into the structural elements important for aminoglycoside inhibitory activities and selectivity for leishmanial cytosolic ribosomes, highlighting a novel synthetic derivative, compound 3: , as a prospective therapeutic candidate for the treatment of VL.
Subject(s)
Antiprotozoal Agents/chemistry , Leishmania/drug effects , Paromomycin/chemistry , Protein Synthesis Inhibitors/chemistry , Ribosomes/drug effects , Animals , Antiprotozoal Agents/pharmacology , Antiprotozoal Agents/toxicity , Binding Sites , Cell Line , Computer Simulation , Guinea Pigs , Humans , Leishmania/growth & development , Macrophages/parasitology , Male , Models, Molecular , Neomycin/analogs & derivatives , Neomycin/chemistry , Neomycin/toxicity , Paromomycin/pharmacology , Paromomycin/toxicity , Protein Biosynthesis/drug effects , Protein Synthesis Inhibitors/pharmacology , Protein Synthesis Inhibitors/toxicity , RNA, Ribosomal/chemistry , Ribosomes/chemistryABSTRACT
The heptameric COPI coat (coatomer) plays an essential role in vesicular transport in the early secretory system of eukaryotic cells. While the structures of some of the subunits have been determined, that of the δ-COP subunit has not been reported to date. The δ-COP subunit is part of a subcomplex with structural similarity to tetrameric clathrin adaptors (APs), where δ-COP is the structural homologue of the AP µ subunit. Here, the crystal structure of the µ homology domain (MHD) of δ-COP (δ-MHD) obtained by phasing using a combined SAD-MR method is presented at 2.15 Å resolution. The crystallographic asymmetric unit contains two monomers that exhibit short sections of disorder, which may allude to flexible regions of the protein. The δ-MHD is composed of two subdomains connected by unstructured linkers. Comparison between this structure and those of known MHD domains from the APs shows significant differences in the positions of specific loops and ß-sheets, as well as a more general change in the relative positions of the protein subdomains. The identified difference may be the major source of cargo-binding specificity. Finally, the crystal structure is used to analyze the potential effect of the I422T mutation in δ-COP previously reported to cause a neurodegenerative phenotype in mice.
Subject(s)
Coat Protein Complex I/chemistry , Animals , Cattle , Crystallography, X-Ray , Mice , Models, Molecular , Mutation , Phenotype , Protein ConformationABSTRACT
Membrane type 1 metalloprotease (MT1-MMP) is a membrane-anchored, zinc-dependent protease. MT1-MMP is an important mediator of cell migration and invasion, and overexpression of this enzyme has been correlated with the malignancy of various tumor types. Therefore, modulators of MT1-MMP activity are proposed to possess therapeutic potential in numerous invasive diseases. Here we report the inhibition mode of MT1-MMP by LEM-2/15 antibody, which targets a surface epitope of MT1-MMP. Specifically, the crystal structures of Fab LEM-2/15 in complex with the MT1-MMP surface antigen suggest that conformational swiveling of the enzyme surface loop is required for effective binding and consequent inhibition of MT1-MMP activity on the cell membrane. This inhibition mechanism appears to be effective in controlling active MT1-MMP in endothelial cells and at the leading edge of migratory cancer cells.
Subject(s)
Antibodies/metabolism , Matrix Metalloproteinase 14/chemistry , Matrix Metalloproteinase 14/metabolism , Matrix Metalloproteinase Inhibitors/metabolism , Animals , Antibodies/chemistry , Catalytic Domain , Cell Membrane/metabolism , Cells, Cultured , Crystallography, X-Ray , Human Umbilical Vein Endothelial Cells , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/metabolism , Matrix Metalloproteinase Inhibitors/chemistry , Models, Molecular , Protein Binding , Protein Structure, QuaternaryABSTRACT
A p53 hot-spot mutation found frequently in human cancer is the replacement of R273 by histidine or cysteine residues resulting in p53 loss of function as a tumor suppressor. These mutants can be reactivated by the incorporation of second-site suppressor mutations. Here, we present high-resolution crystal structures of the p53 core domains of the cancer-related proteins, the rescued proteins and their complexes with DNA. The structures show that inactivation of p53 results from the incapacity of the mutated residues to form stabilizing interactions with the DNA backbone, and that reactivation is achieved through alternative interactions formed by the suppressor mutations. Detailed structural and computational analysis demonstrates that the rescued p53 complexes are not fully restored in terms of DNA structure and its interface with p53. Contrary to our previously studied wild-type (wt) p53-DNA complexes showing non-canonical Hoogsteen A/T base pairs of the DNA helix that lead to local minor-groove narrowing and enhanced electrostatic interactions with p53, the current structures display Watson-Crick base pairs associated with direct or water-mediated hydrogen bonds with p53 at the minor groove. These findings highlight the pivotal role played by R273 residues in supporting the unique geometry of the DNA target and its sequence-specific complex with p53.
Subject(s)
DNA/chemistry , Mutation , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/genetics , Crystallography, X-Ray , Humans , Models, Molecular , Nucleic Acid Conformation , Protein Structure, Tertiary , Suppression, GeneticABSTRACT
Endogenous tissue inhibitors of metalloproteinases (TIMPs) have key roles in regulating physiological and pathological cellular processes. Imitating the inhibitory molecular mechanisms of TIMPs while increasing selectivity has been a challenging but desired approach for antibody-based therapy. TIMPs use hybrid protein-protein interactions to form an energetic bond with the catalytic metal ion, as well as with enzyme surface residues. We used an innovative immunization strategy that exploits aspects of molecular mimicry to produce inhibitory antibodies that show TIMP-like binding mechanisms toward the activated forms of gelatinases (matrix metalloproteinases 2 and 9). Specifically, we immunized mice with a synthetic molecule that mimics the conserved structure of the metalloenzyme catalytic zinc-histidine complex residing within the enzyme active site. This immunization procedure yielded selective function-blocking monoclonal antibodies directed against the catalytic zinc-protein complex and enzyme surface conformational epitopes of endogenous gelatinases. The therapeutic potential of these antibodies has been demonstrated with relevant mouse models of inflammatory bowel disease. Here we propose a general experimental strategy for generating inhibitory antibodies that effectively target the in vivo activity of dysregulated metalloproteinases by mimicking the mechanism employed by TIMPs.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Catalytic Domain/immunology , Inflammatory Bowel Diseases/therapy , Matrix Metalloproteinase Inhibitors , Animals , Antibodies, Monoclonal/immunology , Disease Models, Animal , Female , Humans , Matrix Metalloproteinase 2/chemistry , Matrix Metalloproteinase 2/immunology , Matrix Metalloproteinase 9/chemistry , Matrix Metalloproteinase 9/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Mimicry/immunology , Peptides/chemical synthesis , Peptides/chemistry , Peptides/immunology , Protein Conformation , Tissue Inhibitor of Metalloproteinases/metabolism , Zinc/chemistry , Zinc/immunologyABSTRACT
The structures of the large ribosomal subunit of Deinococcus radiodurans (D50S) in complex with the antibiotic lankamycin (3.2 Å) and a double antibiotic complex of lankamycin and lankacidin C (3.45 Å) have been determined, in continuation of previous crystallographic studies on lankacidin-D50S complex. These two drugs have been previously reported to inhibit ribosomal function with mild synergistic effect. Lankamycin, a member of the macrolide family, binds in a similar manner to erythromycin. However, when in complex with lankacidin, lankamycin is located so that it can form interactions with lankacidin in the adjacent ribosomal binding site. When compared to the well-documented synergistic antibiotics, Streptogramins A and B, the pair of lankacidin and lankamycin bind in similar sites, the peptidyl transferase center and nascent peptide exit tunnel, respectively. Herein, we discuss the structural basis for antibiotic synergism and highlight the key factors involved in ribosomal inhibition.