uu.seUppsala University Publications
Change search
Link to record
Permanent link

Direct link
BETA
Publications (10 of 25) Show all publications
Stsiapanava, A. & Selmer, M. (2019). Crystal structure of ErmE-23S rRNA methyltransferase in macrolide resistance. Scientific Reports, 9(1), Article ID 14607.
Open this publication in new window or tab >>Crystal structure of ErmE-23S rRNA methyltransferase in macrolide resistance
2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, no 1, article id 14607Article in journal (Refereed) Published
Abstract [en]

Pathogens often receive antibiotic resistance genes through horizontal gene transfer from bacteria that produce natural antibiotics. ErmE is a methyltransferase (MTase) from Saccharopolyspora erythraea that dimethylates A2058 in 23S rRNA using S-adenosyl methionine (SAM) as methyl donor, protecting the ribosomes from macrolide binding. To gain insights into the mechanism of macrolide resistance, the crystal structure of ErmE was determined to 1.75 Å resolution. ErmE consists of an N-terminal Rossmann-like α/ß catalytic domain and a C-terminal helical domain. Comparison with ErmC' that despite only 24% sequence identity has the same function, reveals highly similar catalytic domains. Accordingly, superposition with the catalytic domain of ErmC' in complex with SAM suggests that the cofactor binding site is conserved. The two structures mainly differ in the C-terminal domain, which in ErmE contains a longer loop harboring an additional 310 helix that interacts with the catalytic domain to stabilize the tertiary structure. Notably, ErmE also differs from ErmC' by having long disordered extensions at its N- and C-termini. A C-terminal disordered region rich in arginine and glycine is also a present in two other MTases, PikR1 and PikR2, which share about 30% sequence identity with ErmE and methylate the same nucleotide in 23S rRNA.

National Category
Structural Biology
Research subject
Biology with specialization in Structural Biology
Identifiers
urn:nbn:se:uu:diva-395134 (URN)10.1038/s41598-019-51174-0 (DOI)000489555900001 ()
Funder
Swedish Research Council, 2017-03827Swedish Research Council, 2016-06264
Available from: 2019-10-13 Created: 2019-10-13 Last updated: 2019-11-08Bibliographically approved
Jerlström-Hultqvist, J., Warsi, O., Söderholm, A., Knopp, M., Eckhard, U., Vorontsov, E., . . . Andersson, D. I. (2018). A bacteriophage enzyme induces bacterial metabolic perturbation that confers a novel promiscuous function. Nature Ecology & Evolution, 2(8), 1321-1330
Open this publication in new window or tab >>A bacteriophage enzyme induces bacterial metabolic perturbation that confers a novel promiscuous function
Show others...
2018 (English)In: Nature Ecology & Evolution, E-ISSN 2397-334X, Vol. 2, no 8, p. 1321-1330Article in journal (Refereed) Published
Abstract [en]

One key concept in the evolution of new functions is the ability of enzymes to perform promiscuous side-reactions that serve as a source of novelty that may become beneficial under certain conditions. Here, we identify a mechanism where a bacteriophage-encoded enzyme introduces novelty by inducing expression of a promiscuous bacterial enzyme. By screening for bacteriophage DNA that rescued an auxotrophic Escherichia coli mutant carrying a deletion of the ilvA gene, we show that bacteriophage-encoded S-adenosylmethionine (SAM) hydrolases reduce SAM levels. Through this perturbation of bacterial metabolism, expression of the promiscuous bacterial enzyme MetB is increased, which in turn complements the absence of IlvA. These results demonstrate how foreign DNA can increase the metabolic capacity of bacteria, not only by transfer of bona fide new genes, but also by bringing cryptic bacterial functions to light via perturbations of cellular physiology.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Evolutionary Biology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-355286 (URN)10.1038/s41559-018-0568-5 (DOI)000439505600024 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2018-06-27 Created: 2018-06-27 Last updated: 2018-11-22Bibliographically approved
Stern, A. L., Van der Verren, S. E., Kanchugal Puttaswamy, S., Näsvall, J., Gutiérrez-de-Terán, H. & Selmer, M. (2018). Structural mechanism of AadA, a dual specificity aminoglycoside adenylyltransferase from Salmonella enterica. Journal of Biological Chemistry, 293, 11481-11490
Open this publication in new window or tab >>Structural mechanism of AadA, a dual specificity aminoglycoside adenylyltransferase from Salmonella enterica
Show others...
2018 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, p. 11481-11490Article in journal (Refereed) Published
Abstract [en]

Streptomycin and spectinomycin are antibiotics that bind to the bacterial ribosome and perturb protein synthesis. The clinically most prevalent bacterial resistance mechanism is their chemical modification by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). AadA from Salmonella enterica is an aminoglycoside (3’’)(9) adenylyl transferase that O-adenylates position 3” of streptomycin and position 9 of spectinomycin. We previously reported the apo AadA structure with a closed active site. To clarify how AadA binds ATP and its two chemically distinct drug substrates, we here report crystal structures of wildtype AadA complexed with ATP, magnesium, and streptomycin and of an active-site mutant, E87Q, complexed with ATP and streptomycin or the closely related dihydrostreptomycin. These structures revealed that ATP binding induces a conformational change that positions the two domains for drug binding at the interdomain cleft and disclosed the interactions between both domains and the three rings of streptomycin. Spectinomycin docking followed by molecular dynamics simulations suggested that despite the limited structural similarities with streptomycin, spectinomycin makes similar interactions around the modification site, and, in agreement with mutational data, critically interacts with fewer residues. Using structure-guided sequence analyses of ANT(3”)(9) enzymes acting on both substrates and ANT(9) enzymes active only on spectinomycin, we identified sequence determinants for activity on each substrate. We experimentally confirmed that Trp-173 and Asp-178 are essential only for streptomycin resistance. Activity assays indicated that Glu-87 is the catalytic base in AadA and that the non-adenylating E87Q mutant can hydrolyze ATP in the presence of streptomycin.

National Category
Structural Biology Biochemistry and Molecular Biology
Research subject
Biology with specialization in Structural Biology
Identifiers
urn:nbn:se:uu:diva-353761 (URN)10.1074/jbc.RA118.003989 (DOI)000439449700018 ()29871922 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research Council, 2017-03827Swedish Research Council, 2013-05930EU, FP7, Seventh Framework Programme, 283570
Available from: 2018-06-15 Created: 2018-06-15 Last updated: 2020-01-29Bibliographically approved
Newton, M. S., Guo, X., Söderholm, A., Näsvall, J., Lundström, P., Andersson, D. I., . . . Patrick, W. M. (2017). Structural and functional innovations in the real-time evolution of new (βα)8 barrel enzymes. Proceedings of the National Academy of Sciences of the United States of America, 114(8), 4727-4732
Open this publication in new window or tab >>Structural and functional innovations in the real-time evolution of new (βα)8 barrel enzymes
Show others...
2017 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 8, p. 4727-4732Article in journal (Refereed) Published
Abstract [en]

New genes can arise by duplication and divergence, but there is a fundamental gap in our understanding of the relationship between these genes, the evolving proteins they encode, and the fitness of the organism. Here we used crystallography, NMR dynamics, kinetics, and mass spectrometry to explain the molecular innovations that arose during a previous real-time evolution experiment. In that experiment, the (βα)8 barrel enzyme HisA was under selection for two functions (HisA and TrpF), resulting in duplication and divergence of the hisA gene to encode TrpF specialists, HisA specialists, and bifunctional generalists. We found that selection affects enzyme structure and dynamics, and thus substrate preference, simultaneously and sequentially. Bifunctionality is associated with two distinct sets of loop conformations, each essential for one function. We observed two mechanisms for functional specialization: structural stabilization of each loop conformation and substrate-specific adaptation of the active site. Intracellular enzyme performance, calculated as the product of catalytic efficiency and relative expression level, was not linearly related to fitness. Instead, we observed thresholds for each activity above which further improvements in catalytic efficiency had little if any effect on growth rate. Overall, we have shown how beneficial substitutions selected during real-time evolution can lead to manifold changes in enzyme function and bacterial fitness. This work emphasizes the speed at which adaptive evolution can yield enzymes with sufficiently high activities such that they no longer limit the growth of their host organism, and confirms the (βα)8 barrel as an inherently evolvable protein scaffold.

Keywords
HisA, TrpF, adaptive evolution, enzyme performance threshold
National Category
Evolutionary Biology Structural Biology
Research subject
Biology with specialization in Structural Biology; Biochemistry; Biology with specialization in Molecular Evolution
Identifiers
urn:nbn:se:uu:diva-320223 (URN)10.1073/pnas.1618552114 (DOI)000400358000052 ()
Available from: 2017-04-18 Created: 2017-04-18 Last updated: 2018-11-22Bibliographically approved
Ieong, K.-W., Uzun, Ü., Selmer, M. & Ehrenberg, M. (2016). Two proofreading steps amplify the accuracy of genetic code translation. Proceedings of the National Academy of Sciences of the United States of America, 13(48), 13744-13749
Open this publication in new window or tab >>Two proofreading steps amplify the accuracy of genetic code translation
2016 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 13, no 48, p. 13744-13749Article in journal (Refereed) Published
Abstract [en]

Aminoacyl-tRNAs (aa-tRNAs) are selected by the messenger RNA programmed ribosome in ternary complex with elongation factor Tu (EF-Tu) and GTP and then, again, in a proofreading step after GTP hydrolysis on EF-Tu. We use tRNA mutants with different affinities for EF-Tu to demonstrate that proofreading of aatRNAs occurs in two consecutive steps. First, aa-tRNAs in ternary complex with EF-Tu·GDP are selected in a step where the accuracy increases linearly with increasing aa-tRNA affinity to EF-Tu. Then, following dissociation of EF-Tu·GDP from the ribosome, the accuracy is further increased in a second and apparently EFTu−independent step. Our findings identify the molecular basis of proofreading in bacteria, highlight the pivotal role of EF-Tu for fast and accurate protein synthesis, and illustrate the importance of multistep substrate selection in intracellular processing of genetic information.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-235532 (URN)10.1073/pnas.1610917113 (DOI)000388835700066 ()27837019 (PubMedID)
Available from: 2014-11-05 Created: 2014-11-05 Last updated: 2017-12-05Bibliographically approved
Chen, Y., Näsvall, J., Wu, S., Andersson, D. I. & Selmer, M. (2015). Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3'')(9) adenyltransferase. Acta Crystallographica Section D: Biological Crystallography, 71, 2267-2277
Open this publication in new window or tab >>Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3'')(9) adenyltransferase
Show others...
2015 (English)In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 71, p. 2267-2277Article in journal (Refereed) Published
Abstract [en]

Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleo\-tidyltransferases (ANTs). Here, the first crystal structure of an ANT(3$^\prime$$^\prime$)(9) adenyltransferase, AadA from Salmonella enterica, is presented. AadA catalyses the magnesium-dependent transfer of adenosine monophosphate from ATP to the two chemically dissimilar drugs streptomycin and spectinomycin. The structure was solved using selenium SAD phasing and refined to 2.5Å resolution. AadA consists of a nucleotidyltransferase domain and an α-helical bundle domain. AadA crystallizes as a monomer and is a monomer in solution as confirmed by small-angle X-ray scattering, in contrast to structurally similar homodimeric adenylating enzymes such as kanamycin nucleotidyltransferase. Isothermal titration calorimetry experiments show that ATP binding has to occur before binding of the aminoglycoside substrate, and structure analysis suggests that ATP binding repositions the two domains for aminoglycoside binding in the interdomain cleft. Candidate residues for ligand binding and catalysis were subjected to site-directed mutagenesis. In vivo resistance and in vitro binding assays support the role of Glu87 as the catalytic base in adenylation, while Arg192 and Lys205 are shown to be critical for ATP binding.

Keywords
antibiotic resistance, aminoglycoside, X-ray crystallography, small-angle X-ray scattering
National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-265554 (URN)10.1107/S1399004715016429 (DOI)000364553500009 ()
Funder
Swedish Research CouncilSwedish Foundation for Strategic Research Knut and Alice Wallenberg Foundation
Available from: 2015-11-01 Created: 2015-11-01 Last updated: 2017-12-01Bibliographically approved
Söderholm, A., Guo, X., Newton, M. S., Evans, G. B., Näsvall, J., Patrick, W. M. & Selmer, M. (2015). Two-step Ligand Binding in a (βα)8 Barrel Enzyme: Substrate-bound Structures Shed New Light on the Catalytic Cycle of HisA. Journal of Biological Chemistry, 290(41), 24657-24668
Open this publication in new window or tab >>Two-step Ligand Binding in a (βα)8 Barrel Enzyme: Substrate-bound Structures Shed New Light on the Catalytic Cycle of HisA
Show others...
2015 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 41, p. 24657-24668Article in journal (Refereed) Published
Abstract [en]

HisA is a (βα)8 barrel enzyme that catalyzes the Amadori rearrangement of ProFAR to PRFAR in the histidine biosynthesis pathway and it is a paradigm for the study of enzyme evolution. Still, its exact catalytic mechanism has remained unclear. Here, we present crystal structures of wild type Salmonella enterica HisA (SeHisA) in its apo state and of mutants D7N and D7N/D176A in complex with two different conformations of the labile substrate ProFAR, which was structurally visualized for the first time. Site-directed mutagenesis and kinetics demonstrated that Asp7 acts as the catalytic base and Asp176 as the catalytic acid. The SeHisA structures with ProFAR display two different states of the long loops on the catalytic face of the structure, and demonstrate that initial binding of ProFAR to the active site is independent of loop interactions. When the long loops enclose the substrate, ProFAR adopts an extended conformation where its non-reacting half is in a product-like conformation. This change is associated with shifts in a hydrogen-bond network including His47, Asp129, Thr171 and Ser202, all shown to be functionally important. The closed-conformation structure is highly similar to the bi-functional HisA homologue PriA in complex with PRFAR, thus proving that structure and mechanism are conserved between HisA and PriA. This study clarifies the mechanistic cycle of HisA and provides a striking example of how an enzyme and its substrate can undergo coordinated conformational changes before catalysis.

National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-260701 (URN)10.1074/jbc.M115.678086 (DOI)000362598300003 ()26294764 (PubMedID)
Funder
Swedish Research CouncilSwedish Foundation for Strategic Research EU, FP7, Seventh Framework Programme, 283570
Available from: 2015-08-23 Created: 2015-08-23 Last updated: 2018-11-22
Tek, A., Chen, Y., Selmer, M. & Flores, S. C. (2014). Investigating Ribosome Conformations with Multi-Resolution Modeling. Paper presented at 58th Annual Meeting of the Biophysical-Society, FEB 15-19, 2014, San Francisco, CA. Biophysical Journal, 106(2), 491A-491A
Open this publication in new window or tab >>Investigating Ribosome Conformations with Multi-Resolution Modeling
2014 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 2, p. 491A-491AArticle in journal, Meeting abstract (Other academic) Published
National Category
Biophysics
Identifiers
urn:nbn:se:uu:diva-228603 (URN)10.1016/j.bpj.2013.11.2744 (DOI)000337000402717 ()
Conference
58th Annual Meeting of the Biophysical-Society, FEB 15-19, 2014, San Francisco, CA
Available from: 2014-07-18 Created: 2014-07-17 Last updated: 2017-12-05Bibliographically approved
Sun, S., Selmer, M. & Andersson, D. I. (2014). Resistance to beta-Lactam Antibiotics Conferred by Point Mutations in Penicillin-Binding Proteins PBP3, PBP4 and PBP6 in Salmonella enterica. PLoS ONE, 9(5), e97202
Open this publication in new window or tab >>Resistance to beta-Lactam Antibiotics Conferred by Point Mutations in Penicillin-Binding Proteins PBP3, PBP4 and PBP6 in Salmonella enterica
2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 5, p. e97202-Article in journal (Refereed) Published
Abstract [en]

Penicillin-binding proteins (PBPs) are enzymes responsible for the polymerization of the glycan strand and the cross-linking between glycan chains as well as the target proteins for beta-lactam antibiotics. Mutational alterations in PBPs can confer resistance either by reducing binding of the antibiotic to the active site or by evolving a beta-lactamase activity that degrades the antibiotic. As no systematic studies have been performed to examine the potential of all PBPs present in one bacterial species to evolve increased resistance against beta-lactam antibiotics, we explored the ability of fifteen different defined or putative PBPs in Salmonella enterica to acquire increased resistance against penicillin G. We could after mutagenesis and selection in presence of penicillin G isolate mutants with amino-acid substitutions in the PBPs, FtsI, DacB and DacC (corresponding to PBP3, PBP4 and PBP6) with increased resistance against b-lactam antibiotics. Our results suggest that: (i) most evolved PBPs became 'generalists" with increased resistance against several different classes of b-lactam antibiotics, (ii) synergistic interactions between mutations conferring antibiotic resistance are common and (iii) the mechanism of resistance of these mutants could be to make the active site more accessible for water allowing hydrolysis or less binding to b-lactam antibiotics.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-229315 (URN)10.1371/journal.pone.0097202 (DOI)000338213300135 ()
Available from: 2014-08-06 Created: 2014-08-05 Last updated: 2017-12-05Bibliographically approved
Hultqvist, G., Haq, R., Punekar, A., Chi, C., Engström, Å., Bach, A., . . . Jemth, P. (2013). Energetic pathway sampling in a protein interaction domain. Structure, 21, 1193-1202
Open this publication in new window or tab >>Energetic pathway sampling in a protein interaction domain
Show others...
2013 (English)In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 21, p. 1193-1202Article in journal (Other academic) Published
Abstract [en]

The affinity and specificity of protein-ligand interactions are influenced by energeticcrosstalk within the protein domain. However, the molecular details of such intradomain allostery are still unclear. Here, we have experimentally detected and computationally predicted interactionpathways in the postsynaptic density 95/discs large/zonula occludens 1 (PDZ)-peptide ligand model system using wild-type and circularly permuted PDZ proteins. The circular permutant introduced small perturbations in the tertiary structure and a concomitant rewiring of allosteric pathways, allowing us to describe how subtle changes may reshape energetic signaling. The results were analyzed in the context of other members of the PDZ family, which were found to contain distinct interaction pathways for different peptide ligands. The data reveal a fascinating scenario whereby several energetic pathways are sampled within one single domain and distinct pathways are activated by specific protein ligands. 

Keywords
intradomain allostery, PDZ domain, protein binding, circular permutant
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-185579 (URN)10.1016/j.str.2013.05.010 (DOI)000321681600016 ()
Available from: 2012-12-10 Created: 2012-11-26 Last updated: 2017-10-16Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-9079-2774

Search in DiVA

Show all publications