scholarly journals Structural Basis of APH(3′)-IIIa-Mediated Resistance to N1-Substituted Aminoglycoside Antibiotics

2009 ◽  
Vol 53 (7) ◽  
pp. 3049-3055 ◽  
Author(s):  
Desiree H. Fong ◽  
Albert M. Berghuis
Keyword(s):  
Crystal Structure ◽  
Bacterial Resistance ◽  
Aminoglycoside Antibiotics ◽  
Structural Basis ◽  
Amino Group ◽  
Naturally Occurring ◽  
Type Iiia ◽  
Bactericidal Properties ◽  
A Site ◽  
Binding Loop

ABSTRACT Butirosin is unique among the naturally occurring aminoglycosides, having a substituted amino group at position 1 (N1) of the 2-deoxystreptamine ring with an (S)-4-amino-2-hydroxybutyrate (AHB) group. While bacterial resistance to aminoglycosides can be ascribed chiefly to drug inactivation by plasmid-encoded aminoglycoside-modifying enzymes, the presence of an AHB group protects the aminoglycoside from binding to many resistance enzymes, and hence, the antibiotic retains its bactericidal properties. Consequently, several semisynthetic N1-substituted aminoglycosides, such as amikacin, isepamicin, and netilmicin, were developed. Unfortunately, butirosin, amikacin, and isepamicin are not resistant to inactivation by 3′-aminoglycoside O-phosphotransferase type IIIa [APH(3′)-IIIa]. We report here the crystal structure of APH(3′)-IIIa in complex with an ATP analog, AMPPNP [adenosine 5′-(β,γ-imido)triphosphate], and butirosin A to 2.4-Å resolution. The structure shows that butirosin A binds to the enzyme in a manner analogous to other 4,5-disubstituted aminoglycosides, and the flexible antibiotic-binding loop is key to the accommodation of structurally diverse substrates. Based on the crystal structure, we have also constructed a model of APH(3′)-IIIa in complex with amikacin, a commonly used semisynthetic N1-substituted 4,6-disubstituted aminoglycoside. Together, these results suggest a strategy to further derivatize the AHB group in order to generate new aminoglycoside derivatives that can elude inactivation by resistance enzymes while maintaining their ability to bind to the ribosomal A site.

scholarly journals Mechanism of tRNA-mediated +1 ribosomal frameshifting

2018 ◽  
Vol 115 (44) ◽  
pp. 11226-11231 ◽  
Author(s):  
Samuel Hong ◽  
S. Sunita ◽  
Tatsuya Maehigashi ◽  
Eric D. Hoffer ◽  
Jack A. Dunkle ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Elongation Factor ◽  
Amino Acid Sequences ◽  
Structural Basis ◽  
Forward Movement ◽  
Stem Loop ◽  
Bacterial Ribosome ◽  
Conformational Rearrangements ◽  
A Site

Accurate translation of the genetic code is critical to ensure expression of proteins with correct amino acid sequences. Certain tRNAs can cause a shift out of frame (i.e., frameshifting) due to imbalances in tRNA concentrations, lack of tRNA modifications or insertions or deletions in tRNAs (called frameshift suppressors). Here, we determined the structural basis for how frameshift-suppressor tRNASufA6 (a derivative of tRNAPro) reprograms the mRNA frame to translate a 4-nt codon when bound to the bacterial ribosome. After decoding at the aminoacyl (A) site, the crystal structure of the anticodon stem-loop of tRNASufA6 bound in the peptidyl (P) site reveals ASL conformational changes that allow for recoding into the +1 mRNA frame. Furthermore, a crystal structure of full-length tRNASufA6 programmed in the P site shows extensive conformational rearrangements of the 30S head and body domains similar to what is observed in a translocation intermediate state containing elongation factor G (EF-G). The 30S movement positions tRNASufA6 toward the 30S exit (E) site disrupting key 16S rRNA–mRNA interactions that typically define the mRNA frame. In summary, this tRNA-induced 30S domain change in the absence of EF-G causes the ribosome to lose its grip on the mRNA and uncouples the canonical forward movement of the tRNAs during elongation.

scholarly journals Structural basis for EPC1-mediated recruitment of MBTD1 into the NuA4/TIP60 acetyltransferase complex

2019 ◽  
Author(s):  
Heng Zhang ◽  
Maëva Devoucoux ◽  
Xiaosheng Song ◽  
Li Li ◽  
Gamze Ayaz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Crystal Structure ◽  
Dna Repair ◽  
Structural Basis ◽  
Repair Pathway ◽  
Dna Breaks ◽  
Cellular Assays ◽  
Structural Framework ◽  
A Site ◽  
Key Residues

SummaryMBTD1, a H4K20me reader, has recently been identified as a component of the NuA4/TIP60 acetyltransferase complex, regulating gene expression and DNA repair. NuA4/TIP60 inhibits 53BP1 binding to chromatin through recognition of the H4K20me mark by MBTD1 and acetylation of H2AK15, blocking the ubiquitination mark required for 53BP1 localization at DNA breaks. The NuA4/TIP60 non-catalytic subunit EPC1 enlists MBTD1 into the complex, but the detailed molecular mechanism remains incompletely explored. Here, we present the crystal structure of the MBTD1-EPC1 complex, revealing a hydrophobic C-terminal fragment of EPC1 engaging the MBT repeats of MBTD1 in a site distinct from the H4K20me binding site. Different cellular assays validate the physiological significance of the key residues involved in the MBTD1-EPC1 interaction. Our study provides a structural framework for understanding the mechanism by which MBTD1 recruits the NuA4/TIP60 acetyltransferase complex to influence transcription and DNA repair pathway choice.

scholarly journals Crystal Structure of the Mycobacterium fortuitum Class A β-Lactamase: Structural Basis for Broad Substrate Specificity

2006 ◽  
Vol 50 (7) ◽  
pp. 2516-2521 ◽  
Author(s):  
Eric Sauvage ◽  
Eveline Fonzé ◽  
Birgit Quinting ◽  
Moreno Galleni ◽  
Jean-Marie Frère ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Broad Spectrum ◽  
Active Site ◽  
Bacterial Resistance ◽  
Specific Activity ◽  
Structural Basis ◽  
Mycobacterium Fortuitum ◽  
Bacterial Strains ◽  
Class A ◽  
Extended Spectrum

ABSTRACT β-Lactamases are the main cause of bacterial resistance to penicillins and cephalosporins. Class A β-lactamases, the largest group of β-lactamases, have been found in many bacterial strains, including mycobacteria, for which no β-lactamase structure has been previously reported. The crystal structure of the class A β-lactamase from Mycobacterium fortuitum (MFO) has been solved at 2.13-Å resolution. The enzyme is a chromosomally encoded broad-spectrum β-lactamase with low specific activity on cefotaxime. Specific features of the active site of the class A β-lactamase from M. fortuitum are consistent with its specificity profile. Arg278 and Ser237 favor cephalosporinase activity and could explain its broad substrate activity. The MFO active site presents similarities with the CTX-M type extended-spectrum β-lactamases but lacks a specific feature of these enzymes, the VNYN motif (residues 103 to 106), which confers on CTX-M-type extended-spectrum β-lactamases a more efficient cefotaximase activity.

scholarly journals Binding of Neomycin-Class Aminoglycoside Antibiotics to Mutant Ribosomes with Alterations in the A Site of 16S rRNA

2006 ◽  
Vol 50 (4) ◽  
pp. 1489-1496 ◽  
Author(s):  
Sven N. Hobbie ◽  
Peter Pfister ◽  
Christian Bruell ◽  
Peter Sander ◽  
Boris François ◽  
...  
Keyword(s):  
Crystal Structure ◽  
16S Rrna ◽  
Base Pair ◽  
Mycobacterium Smegmatis ◽  
Drug Binding ◽  
Aminoglycoside Antibiotics ◽  
Wild Type ◽  
The Core ◽  
A Site

ABSTRACT Aminoglycoside antibiotics that bind to the aminoacyl-tRNA site (A site) of the ribosome are composed of a common neamine core in which a glycopyranosyl ring is attached to position 4 of a 2-deoxystreptamine moiety. The core is further substituted by one (ribostamycin), two (neomycin and paromomycin), or three (lividomycin A) additional sugars attached to position 5 of the 2-deoxystreptamine. To study the role of rings III, IV, and V in aminoglycoside binding, we used isogenic Mycobacterium smegmatis ΔrrnB mutants carrying homogeneous populations of mutant ribosomes with alterations in the 16S rRNA A site. MICs were determined to investigate drug-ribosome interactions, and the results were compared with that of the previously published crystal structure of paromomycin bound to the ribosomal A site. Our analysis demonstrates that the stacking interaction between ring I and G1491 is largely sequence independent, that rings III and IV each increase the strength of drug binding to the ribosome, that ring IV of the 6′-NH3 + aminoglycosides compensates for loss of interactions between ring II and U1495 and between ring III and G1491, that the aminoglycosides rely on pseudo-base pairing between ring I and A1408 for binding independently of the number of sugar rings attached to the neamine core, that addition of ring V to the 6′-OH 4,5-aminoglycoside paromomycin does not alter the mode of binding, and that alteration of the U1406 · U1495 wobble base pair to the Watson-Crick interaction pair 1406C-1495G yields ribosomal drug susceptibilities to 4,5-aminoglycosides comparable to those seen with the wild-type A site.

Recognition of aminoglycoside antibiotics by enterococcal-staphylococcal aminoglycoside 3'-phosphotransferase type IIIa: role of substrate amino groups.

1996 ◽  
Vol 40 (11) ◽  
pp. 2648-2650 ◽  
Author(s):  
G A McKay ◽  
J Roestamadji ◽  
S Mobashery ◽  
G D Wright
Keyword(s):  
Steady State ◽  
Aminoglycoside Antibiotics ◽  
Hydroxyl Group ◽  
Amino Group ◽  
Amino Groups ◽  
Type Iiia ◽  
Steady State Kinetic ◽  
State Kinetic

The interactions of the aminoglycoside 3'-phosphotransferase IIIa with aminoglycoside antibiotics lacking specific amino groups were examined by steady-state kinetic analyses. The results demonstrate that an amino group on C-1 and either an amino or a hydroxyl group at the 2' and 6' positions are important for detoxification of aminoglycosides by this enzyme.

scholarly journals Structural basis of Naa20 activity towards a canonical NatB substrate

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Dominik Layer ◽  
Jürgen Kopp ◽  
Miriam Fontanillo ◽  
Maja Köhn ◽  
Karine Lapouge ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Drug Development ◽  
Catalytic Mechanism ◽  
Peptide Binding ◽  
Competitive Inhibitor ◽  
Structural Basis ◽  
Substrate Affinity ◽  
Protein Homeostasis ◽  
Auxiliary Subunit

AbstractN-terminal acetylation is one of the most common protein modifications in eukaryotes and is carried out by N-terminal acetyltransferases (NATs). It plays important roles in protein homeostasis, localization, and interactions and is linked to various human diseases. NatB, one of the major co-translationally active NATs, is composed of the catalytic subunit Naa20 and the auxiliary subunit Naa25, and acetylates about 20% of the proteome. Here we show that NatB substrate specificity and catalytic mechanism are conserved among eukaryotes, and that Naa20 alone is able to acetylate NatB substrates in vitro. We show that Naa25 increases the Naa20 substrate affinity, and identify residues important for peptide binding and acetylation activity. We present the first Naa20 crystal structure in complex with the competitive inhibitor CoA-Ac-MDEL. Our findings demonstrate how Naa20 binds its substrates in the absence of Naa25 and support prospective endeavors to derive specific NAT inhibitors for drug development.

scholarly journals Crystal structures of Ca2+–calmodulin bound to NaV C-terminal regions suggest role for EF-hand domain in binding and inactivation

2019 ◽  
Vol 116 (22) ◽  
pp. 10763-10772 ◽  
Author(s):  
Bernd R. Gardill ◽  
Ricardo E. Rivera-Acevedo ◽  
Ching-Chieh Tung ◽  
Filip Van Petegem
Keyword(s):  
Crystal Structure ◽  
Binding Sites ◽  
Binding Mode ◽  
Conformational Switch ◽  
Channel Inactivation ◽  
Dependent Inactivation ◽  
Ef Hand ◽  
Inherited Arrhythmia ◽  
A Site

Voltage-gated sodium (NaV) and calcium channels (CaV) form targets for calmodulin (CaM), which affects channel inactivation properties. A major interaction site for CaM resides in the C-terminal (CT) region, consisting of an IQ domain downstream of an EF-hand domain. We present a crystal structure of fully Ca2+-occupied CaM, bound to the CT of NaV1.5. The structure shows that the C-terminal lobe binds to a site ∼90° rotated relative to a previous site reported for an apoCaM complex with the NaV1.5 CT and for ternary complexes containing fibroblast growth factor homologous factors (FHF). We show that the binding of FHFs forces the EF-hand domain in a conformation that does not allow binding of the Ca2+-occupied C-lobe of CaM. These observations highlight the central role of the EF-hand domain in modulating the binding mode of CaM. The binding sites for Ca2+-free and Ca2+-occupied CaM contain targets for mutations linked to long-QT syndrome, a type of inherited arrhythmia. The related NaV1.4 channel has been shown to undergo Ca2+-dependent inactivation (CDI) akin to CaVs. We present a crystal structure of Ca2+/CaM bound to the NaV1.4 IQ domain, which shows a binding mode that would clash with the EF-hand domain. We postulate the relative reorientation of the EF-hand domain and the IQ domain as a possible conformational switch that underlies CDI.

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