Biology subjects

Grassucci, R. A.

Publications and source records attributed to Grassucci, R. A..

3 recordsLinked to original sources

bioRxiv2023

Cryo-EM studies of the four E. coli paralogs establish ABCF proteins as master plumbers of the peptidyl-transferase center of the ribosome

The genomes of most mesophilic organisms encode multiple ATP-Binding Cassette F (ABCF) proteins. EttA, one of four E. coli paralogs, regulates synthesis of the first peptide bond on the ribosome dependent on ATP/ADP ratio, while Antibiotic Resistance factors (AREs), paralogs in other organisms, both regulate and directly mediate resistance to ribosome-targeted antibiotics. However, the physiological functions remain unclear for most paralogs, and the mechanism-of-action has yet to be rigorously established for any paralog. We herein present single particle cryogenic electron microscopy structures of ribosome complexes of all four E. coli ABCF paralogs (EttA, Uup, YbiT, and YheS), which, together with previously determined ARE structures, show that ABCFs control the binding geometry of the tRNA in the peptidyl-tRNA-binding (P) site on the ribosome. They modulate the position of its acceptor stem relative to the peptidyl transferase center (PTC) in a manner that can either promote (EttA and Uup) or disrupt (YbiT, YheS, and the AREs) proper catalytic geometry. The YbiT/70S reconstructions include a conformation with no density for ribosomal protein bL33, and structural analyses support the exchange of this sub-stoichiometric ribosomal protein being functionally related to conformational changes in YbiT controlled by sequence variations in the strongly non-canonical Signature Sequence in its first ABC domain. Our studies establish general structural/enzymological principles by which the ATPase activity of ABCF proteins controls translation elongation coupled to modulation of conformation and stereochemistry in the catalytic core of the ribosome.

biophysics↗

bioRxiv2022

Mechanism of dual pharmacological correction and potentiation of human CFTR

Cystic fibrosis (CF) is caused by mutations in a chloride channel called the human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR). We used cryo-EM global conformational ensemble reconstruction to characterize the mechanism by which the breakthrough drug VX445 (Elexacaftor) simultaneously corrects both protein-folding and channel-gating defects caused by CF mutations. VX445 drives hCFTR molecules harboring the gating-defective G551D mutation towards the open-channel conformation by binding to a site in the first transmembrane domain. This binding interaction reverses the usual pathway of allosteric structural communication by which ATP binding activates channel conductance, which is blocked by the G551D mutation. Our ensemble reconstructions include a 3.4 [A] non-native structure demonstrating that detachment of the first nucleotide-binding domain of hCFTR is directly coupled to local unfolding of the VX445 binding site. Reversal of this unfolding transition likely contributes to its corrector activity by cooperatively stabilizing NBD1 and the transmembrane domains of hCFTR during biogenesis. SummaryCryo-EM global conformational ensemble reconstruction has been used to characterize the mechanism-of-action of a breakthrough pharmaceutical that corrects fatal protein-folding and channel-gating defects in the human cystic fibrosis transmembrane conductance regulator (CFTR).

biophysics↗

bioRxiv2018

The structural basis for release factor activation during translation termination revealed by time-resolved cryogenic electron microscopy

When the mRNA translating ribosome encounters a stop codon in its aminoacyl site (A site), it recruits a class-1 release factor (RF) to induce hydrolysis of the ester bond between peptide chain and peptidyl-site (P-site) tRNA. This process, called termination of translation, is under strong selection pressure for high speed and accuracy. Class-1 RFs (RF1, RF2 in bacteria, eRF1 in eukarya and aRF1 in archaea), have structural motifs that recognize stop codons in the decoding center (DC) and a universal GGQ motif for induction of ester bond hydrolysis in the peptidyl transfer center (PTC) 70 [A] away from the DC. The finding that free RF2 is compact with only 20 [A] between its codon reading and GGQ motifs came therefore as a surprise1. Cryo-electron microscopy (cryo-EM) then showed that ribosome-bound RF1 and RF2 have extended structures2,3, suggesting that bacterial RFs are compact when entering the ribosome and switch to the extended form in a stop signal-dependent manner3. FRET4, cryo-EM5,6 and X-ray crystallography7, along with a rapid kinetics study suggesting a pre-termination conformational change on the millisecond time-scale of ribosome-bound RF1 and RF28, have lent indirect support to this proposal. However, direct experimental evidence for such a short-lived compact conformation on the native pathway to RF-dependent termination is missing due to its transient nature. Here we use time-resolved cryo-EM9,10,11,12,13 to visualize compact and extended forms of RF1 and RF2 at 3.5 and 4 [A] resolution, respectively, in the codon-recognizing complex on the pathway to termination. About 25% of ribosomal complexes have RFs in the compact state at 24 ms reaction time after mixing RF and ribosomes, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms.

biophysics↗