Steven R. Herron

The role of SmpB and the ribosomal decoding center in licensing tmRNA entry into stalled ribosomes

In bacteria, stalled ribosomes are recycled by a hybrid transfer-messenger RNA (tmRNA). Like tRNA, tmRNA is aminoacylated with alanine and is delivered to the ribosome by EF-Tu, where it reacts with the growing polypeptide chain. tmRNA entry into stalled ribosomes poses a challenge to our understanding of ribosome function because it occurs in the absence of a codon-anticodon interaction. Instead, tmRNA entry is licensed by the binding of its protein partner, SmpB, to the ribosomal decoding center. We analyzed a series of SmpB mutants and found that its C-terminal tail is essential for tmRNA accommodation but not for EF-Tu activation. We obtained evidence that the tail likely functions as a helix on the ribosome to promote accommodation and identified key residues in the tail essential for this step. In addition, our mutational analysis points to a role for the conserved K(131)GKK tail residues in trans-translation after peptidyl transfer to tmRNA, presumably EF-G-mediated translocation or translation of the tmRNA template. Surprisingly, analysis of A1492, A1493, and G530 mutants reveals that while these ribosomal nucleotides are essential for normal tRNA selection, they play little to no role in peptidyl transfer to tmRNA. These studies clarify how SmpB interacts with the ribosomal decoding center to license tmRNA entry into stalled ribosomes.

Inhibition of Aminoglycoside 6′-N-Acetyltransferase Type Ib by Zinc: Reversal of Amikacin Resistance in Acinetobacter baumannii and Escherichia coli by a Zinc Ionophore

ABSTRACT In vitro activity of the aminoglycoside 6′-N-acetyltransferase type Ib [AAC(6′)-Ib] was inhibited by ZnCl2 with a 50% inhibitory concentration (IC50) of 15 μM. Growth of Acinetobacter baumannii or Escherichia coli harboring aac(6′)-Ib in cultures containing 8 μg/ml amikacin was significantly inhibited by the addition of 2 μM Zn2+ in complex with the ionophore pyrithione (ZnPT).

High resolution crystal structure of ferricytochrome c' from Rhodobacter sphaeroides

Cytochrome c′ isolated from Rhodobacter sphaeroides strain R26 (RSCP) crystallizes as a dimer of two identical 14-kDa subunits, in trigonal space group P31, with cell parameters a, b = 48.10 Å, c = 115.80 Å. The crystal structure of RSCP has been solved by molecular replacement using cytochrome c′ from Rhodobacter capsulatus (PDB ID: 1CPQ) as a search model. To ensure effective phase bias removal, the RSCP model was iteratively built into maps generated by a modified wARP procedure, Shake&wARP. The 1.8 Å model (PDB ID: 1GQA) has been refined to an R = 0.204 and freeR = 0.254. Each subunit consists of four antiparallel α-helices, with the pentacoordinate heme covalently bound to a C–X–Y–C–H motif near the C-terminus. F14, located on helix A, blocks direct access to what would be the sixth “distal” ligand binding site of the heme. The dimer subunits form a flattened “X” shape, intermediate between the Type 1 and Type 2 cytochromes c′. The presence of the aromatic F14 and a deep channel between helices B and C places RSCP into Group 1 cytochromes c′. Clear electron density has revealed that the amino acid sequences for the cytochrome c′ from strains R26 and 2.4.1 are identical.

Crystal structures of diphosphinated group 6 Fischer alkoxy carbenes

The crystal structures of four diphosphinated Group 6 Fischer alkoxy carbenes with the compositions fac-(dppe)(CO)3M–C(OR′)(R) (R = Me, R′ = Et, M = Cr, 1; R = Ph, R′ = Me, M = Cr, 2; R = Me, R′ = Me, M = W, 3; R/OR′ = 3-methyl-2-oxacyclopentylidenyl, M = Cr, 4) have been determined at 243 K. Compound 1 crystallizes in the monoclinic system, space group P21/c with a = 11.2243(11) Å, b = 18.5998(18) Å, c = 15.1260(15) Å, β = 107.056(4)○, V = 3019.0(5) Å3, and Z = 4. Compound 2 crystallizes in the monoclinic system, space group P21/c with a = 11.8102(9) Å, b = 18.3152(14) Å, c = 15.0262(12) Å, β = 93.753(3)○, V = 3243.3(4) Å3, and Z = 4. Compound 3 crystallizes in the monoclinic system, space group P21/c with a = 11.3458(6) Å, b = 18.5772(9) Å, c = 15.3883(8) Å, β = 108.576(6)○, V = 3074.5(3) Å3, and Z = 4. Compound 4 crystallizes in the orthorhombic system, space group Pna21 with a = 22.6509(14) Å, b = 9.8118(6) Å, c = 13.7507(8) Å, V = 3056.0(3) Å3, and Z = 4. Steric repulsions with the dppe ligand favor a conformation with the alkoxy moiety directed toward the dppe backbone, in an E geometry, in 1–4.

Synthesis, reactivity and structural study of diastereomeric titanium complexes with amino acid derived N- and O-π donor ligands

Abstract Two diastereomeric Ti(IV) complexes of the type [Ti(Cl)(NMe 2 )(OCH 2 CH(CH 2 Ph)N(R))] 2 (where R= i Pr or cyclo -C 6 H 11 ) have been synthesized by protonolysis of TiCl(NMe 2 ) 3 with the corresponding N-substituted amino alcohols. The chiral ligands were synthesized in a two-step procedure from l -phenylalanine ethyl ester. The complexes bridge through the amino alcohol oxygen atoms, and contain terminal chlorides. The remaining dimethylamide group can be replaced by a protonolysis reaction with 2,6-diisopropylphenol. The bulky disubstituted phenoxide ligand does not inhibit dimerization, and the amino alcohol oxygen bridged complex [Ti(Cl)( O -2,6- i Pr 2 C 6 H 3 )(OCH 2 CH(CH 2 Ph)NR)] 2 is obtained. The dimeric nature of the complexes was established by X-ray crystallography.

Pyridine-pyrimidine amides that prevent HGF-induced epithelial scattering by two distinct mechanisms

Stimulation of cultured epithelial cells with scatter factor/hepatocyte growth factor (HGF) results in individual cells detaching and assuming a migratory and invasive phenotype. Epithelial scattering recapitulates cancer progression and studies have implicated HGF signaling as a driver of cancer metastasis. Inhibitors of HGF signaling have been proposed to act as anti-cancer agents. We previously screened a small molecule library for compounds that block HGF-induced epithelial scattering. Most hits identified in this screen exhibit anti-mitotic properties. Here we assess the biological mechanism of a compound that blocks HGF-induced scattering with limited anti-mitotic activity. Analogs of this compound have one of two distinct activities: inhibiting either cell migration or cell proliferation with cell cycle arrest in G2/M. Each activity bears unique structure-activity relationships. The mechanism of action of anti-mitotic compounds is by inhibition of microtubule polymerization; these compounds entropically and enthalpically bind tubulin in the colchicine binding site, generating a conformational change in the tubulin dimer.