Miri Krupkin

All Publications

• Avilamycin and evernimicin induce structural changes in rProteins uL16 and CTC that enhance the inhibition of A-site tRNA binding. Proceedings of the National Academy of Sciences of the United States of America Krupkin, M., Wekselman, I., Matzov, D., Eyal, Z., Diskin Posner, Y., Rozenberg, H., Zimmerman, E., Bashan, A., Yonath, A. 2016; 113 (44): E6796–E6805

  Abstract

  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.

  View details for DOI 10.1073/pnas.1614297113

  View details for PubMedID 27791159

  View details for PubMedCentralID PMC5098648

• A vestige of a prebiotic bonding machine is functioning within the contemporary ribosome. Philosophical transactions of the Royal Society of London. Series B, Biological sciences Krupkin, M., Matzov, D., Tang, H., Metz, M., Kalaora, R., Belousoff, M. J., Zimmerman, E., Bashan, A., Yonath, A. 2011; 366 (1580): 2972–78

  Abstract

  Based on the presumed capability of a prebiotic pocket-like entity to accommodate substrates whose stereochemistry enables the creation of chemical bonds, it is suggested that a universal symmetrical region identified within all contemporary ribosomes originated from an entity that we term the 'proto-ribosome'. This 'proto-ribosome' could have evolved from an earlier machine that was capable of performing essential tasks in the RNA world, called here the 'pre-proto-ribosome', which was adapted for producing proteins.

  View details for DOI 10.1098/rstb.2011.0146

  View details for PubMedID 21930590

  View details for PubMedCentralID PMC3158926

• Structural insights of lincosamides targeting the ribosome of Staphylococcus aureus. Nucleic acids research Matzov, D., Eyal, Z., Benhamou, R. I., Shalev-Benami, M., Halfon, Y., Krupkin, M., Zimmerman, E., Rozenberg, H., Bashan, A., Fridman, M., Yonath, A. 2017; 45 (17): 10284–92

  Abstract

  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.

  View details for DOI 10.1093/nar/gkx658

  View details for PubMedID 28973455

  View details for PubMedCentralID PMC5622323

• The Ribosomal Protein uL22 Modulates the Shape of the Protein Exit Tunnel. Structure (London, England : 1993) Wekselman, I., Zimmerman, E., Davidovich, C., Belousoff, M., Matzov, D., Krupkin, M., Rozenberg, H., Bashan, A., Friedlander, G., Kjeldgaard, J., Ingmer, H., Lindahl, L., Zengel, J. M., Yonath, A. 2017; 25 (8): 1233–41.e3

  Abstract

  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 β 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 β hairpin of uL22 that renders resistance to erythromycin. The structure reveals a shift of the β 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.

  View details for DOI 10.1016/j.str.2017.06.004

  View details for PubMedID 28689968

• A novel pleuromutilin antibacterial compound, its binding mode and selectivity mechanism. Scientific reports Eyal, Z., Matzov, D., Krupkin, M., Paukner, S., Riedl, R., Rozenberg, H., Zimmerman, E., Bashan, A., Yonath, A. 2016; 6: 39004

  Abstract

  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.

  View details for DOI 10.1038/srep39004

  View details for PubMedID 27958389

  View details for PubMedCentralID PMC5154188

• Ribosomal Antibiotics: Contemporary Challenges. Antibiotics (Basel, Switzerland) Auerbach-Nevo, T., Baram, D., Bashan, A., Belousoff, M., Breiner, E., Davidovich, C., Cimicata, G., Eyal, Z., Halfon, Y., Krupkin, M., Matzov, D., Metz, M., Rufayda, M., Peretz, M., Pick, O., Pyetan, E., Rozenberg, H., Shalev-Benami, M., Wekselman, I., Zarivach, R., Zimmerman, E., Assis, N., Bloch, J., Israeli, H., Kalaora, R., Lim, L., Sade-Falk, O., Shapira, T., Taha-Salaime, L., Tang, H., Yonath, A. 2016; 5 (3)

  Abstract

  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.

  View details for DOI 10.3390/antibiotics5030024

  View details for PubMedID 27367739

  View details for PubMedCentralID PMC5039520

• A Recombinant Collagen-mRNA Platform for Controllable Protein Synthesis. Chembiochem : a European journal of chemical biology Sun, L., Xiong, Y., Bashan, A., Zimmerman, E., Shulman Daube, S., Peleg, Y., Albeck, S., Unger, T., Yonath, H., Krupkin, M., Matzov, D., Yonath, A. 2015; 16 (10): 1415–19

  Abstract

  We have developed a collagen-mRNA platform for controllable protein production that is intended to be less prone to the problems associated with commonly used mRNA therapy as well as with collagen skin-healing procedures. A collagen mimic was constructed according to a recombinant method and was used as scaffold for translating mRNA chains into proteins. Cysteines were genetically inserted into the collagen chain at positions allowing efficient ribosome translation activity while minimizing mRNA misfolding and degradation. Enhanced green fluorescence protein (eGFP) mRNA bound to collagen was successfully translated by cell-free Escherichia coli ribosomes. This system enabled an accurate control of specific protein synthesis by monitoring expression time and level. Luciferase-mRNA was also translated on collagen scaffold by eukaryotic cell extracts. Thus we have demonstrated the feasibility of controllable protein synthesis on collagen scaffolds by ribosomal machinery.

  View details for DOI 10.1002/cbic.201500205

  View details for PubMedID 25930950

  View details for PubMedCentralID PMC4517095

• Structural insights into species-specific features of the ribosome from the pathogen Staphylococcus aureus. Proceedings of the National Academy of Sciences of the United States of America Eyal, Z., Matzov, D., Krupkin, M., Wekselman, I., Paukner, S., Zimmerman, E., Rozenberg, H., Bashan, A., Yonath, A. 2015; 112 (43): E5805–14

  Abstract

  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.

  View details for DOI 10.1073/pnas.1517952112

  View details for PubMedID 26464510

  View details for PubMedCentralID PMC4629319

• Protoribosome by quantum kernel energy method. Proceedings of the National Academy of Sciences of the United States of America Huang, L., Krupkin, M., Bashan, A., Yonath, A., Massa, L. 2013; 110 (37): 14900–14905

  Abstract

  Experimental evidence suggests the existence of an RNA molecular prebiotic entity, called by us the "protoribosome," which may have evolved in the RNA world before evolution of the genetic code and proteins. This vestige of the RNA world, which possesses all of the capabilities required for peptide bond formation, seems to be still functioning in the heart of all of the contemporary ribosome. Within the modern ribosome this remnant includes the peptidyl transferase center. Its highly conserved nucleotide sequence is suggestive of its robustness under diverse environmental conditions, and hence on its prebiotic origin. Its twofold pseudosymmetry suggests that this entity could have been a dimer of self-folding RNA units that formed a pocket within which two activated amino acids might be accommodated, similar to the binding mode of modern tRNA molecules that carry amino acids or peptidyl moieties. Using quantum mechanics and crystal coordinates, this work studies the question of whether the putative protoribosome has properties necessary to function as an evolutionary precursor to the modern ribosome. The quantum model used in the calculations is density functional theory--B3LYP/3-21G*, implemented using the kernel energy method to make the computations practical and efficient. It occurs that the necessary conditions that would characterize a practicable protoribosome--namely (i) energetic structural stability and (ii) energetically stable attachment to substrates--are both well satisfied.

  View details for DOI 10.1073/pnas.1314112110

  View details for PubMedID 23980159

  View details for PubMedCentralID PMC3773780

• The Proto-Ribosome: an ancient nano-machine for peptide bond formation. Israel journal of chemistry Davidovich, C., Belousoff, M., Wekselman, I., Shapira, T., Krupkin, M., Zimmerman, E., Bashan, A., Yonath, A. 2010; 50 (1): 29–35

  Abstract

  The ribosome is a ribozyme whose active site, the peptidyl transferase center (PTC) is situated within a highly conserved universal symmetrical region that connects all ribosomal functional centers involved in amino-acid polymerization. The linkage between this elaborate architecture and A-site tRNA position revealed that the A to P-site passage of the tRNA 3' terminus during protein synthesis is performed by a rotary motion, synchronized with the overall tRNA/mRNA sideways movement and Guided by the PTC. This rotary motion leads to suitable stereochemistry for peptide bond formation as well as for substrate mediated catalysis. Analysis of the substrate binding modes to ribosomes led to the hypothesis that the ancient ribosome produced single peptide bonds and non-coded chains, potentially in a similar manner to the modern PTC. Later in evolution, a mechanism, enabling some type of decoding genetic control triggered the emergence of the small ribosomal subunit or part of it. This seems to be the result of the appearance of reaction products that could have evolved after polypeptides capable of enzymatic function were generated sporadically, while an ancient stable RNA fold was converted into an old version of a tRNA molecule. As in the contemporary ribosome the symmetry relates only the backbone fold and nucleotides orientations but not nucleotide sequences, it emphasizes the superiority of functional requirement over sequence conservation, and indicates that the PTC may have evolved by gene fusion or gene duplication.

  View details for DOI 10.1002/ijch.201000012

  View details for PubMedID 26207070

  View details for PubMedCentralID PMC4508870