Biology subjects

Majumdar, C.

Publications and source records attributed to Majumdar, C..

3 recordsLinked to original sources

bioRxiv2026

Enzymatic formation of a conserved isoaspartate in ribosomal protein uS11

Isoaspartate (isoAsp) formation is typically viewed as a "molecular clock" through nonenzymatic degradation of aspartate or asparagine during protein aging. Here we report a nearly universal enzymatic pathway for the formation of a conserved isoAsp in the bacterial ribosomal protein uS11. Proteome-wide protein-protein interaction scans using AlphaFold3 identified YbeY as a candidate enzyme from Escherichia coli. NMR spectroscopy supported a stable YbeY-uS11 complex from Thermotoga maritima. Biochemical assays indicated that EcYbeY catalysis is zinc-dependent and prefers the conserved Asn-Gly motif for isoAsp formation. A high-resolution cryo-electron microscopy structure of the 70S ribosome from E. coli {Delta}ybeY revealed that loss of isoAsp alters contacts with the 16S rRNA groove and bS21. Phylogenetic analysis indicated that YbeY is present in almost all bacteria, and its absence is correlated to changes in the Asn-Gly motif of uS11. Additionally, our structural analyses implicate Fap7 as the functional counterpart in archaea and eukaryotes.

biochemistry↗

bioRxiv2026

Altering the ribosome exit tunnel to improve consecutive incorporation of challenging monomers

Ribosomes are capable of incorporating a wide array of natural and unnatural monomers into growing polymer chains, but can be stalled by monomers with constrained or non-natural backbones. Here we evaluate whether monomer-dependent ribosome stalling can be alleviated by structure-guided mutations to 23S rRNA within the exit tunnel. Ribosomes harboring an A2062U mutation are as active as wild type (WT) ribosomes when translating non-proline sequences and up to 10-fold more active when translating sequences containing up to four consecutive proline residues. High-resolution cryo-EM structures of WT and A2062U mutant ribosomes containing a polyproline nascent chain reveal that the A2062U mutation relieves an exit tunnel constriction to better accommodate a conformationally restricted peptide chain. A2062U mutant ribosomes also improve translation of sequences containing multiple, consecutive {beta}2-hydroxy acids. These results provide a mechanistic basis for engineering the ribosome for improved translation of genetically encoded polymers with novel backbones.

biochemistry↗

bioRxiv2023

Aminobenzoic acid derivatives obstruct induced fit in the catalytic center of the ribosome

The Escherichia coli ribosome can incorporate a variety of non-O_SCPLOWLC_SCPLOW--amino acid monomers into polypeptide chains, but with poor efficiency. Although these monomers span a diverse set of compounds, there exists no high-resolution structural information regarding their positioning within the catalytic center of the ribosome, the peptidyl transferase center (PTC). Thus, details regarding the mechanism of amide bond formation and the structural basis for differences and defects in incorporation efficiency remain unknown. Within a set of three aminobenzoic acid derivatives-3-aminopyridine-4-carboxylic acid (Apy), ortho-aminobenzoic acid (oABZ), and meta-aminobenzoic acid (mABZ)-the ribosome incorporates Apy into polypeptide chains with the highest efficiency, followed by oABZ and then mABZ, a trend that does not track with the nucleophilicity of the reactive amines. Here, we report high resolution cryo-EM structures of the ribosome with these three aminobenzoic acid derivatives charged on tRNA bound in the aminoacyl-tRNA site (A site). These structures reveal how the aromatic ring of each monomer sterically blocks positioning of nucleotide U2506, thereby preventing rearrangement of nucleotide U2585 and the resulting induced fit in the PTC required for efficient amide bond formation. They also reveal disruptions to the "proton wire" responsible for facilitating formation and breakdown of the tetrahedral intermediate. Together, the cryo-EM structures reported here provide a clear rationale for differences in reactivity of aminobenzoic acid derivatives relative to O_SCPLOWLC_SCPLOW--amino acids and each other, and point to stereochemical constraints on the size and geometry of non-proteinogenic monomers that can be accepted efficiently by wild-type ribosomes.

biophysics↗