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 ΔybeY 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. Table of Contents
Yanqing Xue, Chandrima Majumdar, Salimat O. Sofela et al.· bioRxiv· 0 citations
Spns transporters are a mechanistically distinct branch of the major facilitator superfamily that regulate lipid transport, lysosomal homeostasis, immunity and disease, yet how the conserved Spns fold integrates protonation, substrate binding and alternating access to support chemically and directionally diverse transport activities remains unresolved. Here, we combine DEER spectroscopy in lipid nanodiscs with DEER- and AlphaFold-guided modeling to define the conformational landscape of the Mycobacterium smegmatis homolog MsSpns. Protonation shifts MsSpns toward an inward-facing state, whereas deprotonation favors a broader outward-facing ensemble through coordinated remodeling of intracellular and extracellular gates. These transitions are governed by membrane-embedded protonation switches and proton-sensing networks, while the substrate-binding cavity shows distinct proton sensitivity and weaker cooperativity. Hydrophilic cationic substrates, capreomycin and ethidium bromide, stabilize the outward-facing state, consistent with efflux antiport, whereas lipophilic compounds, including rifampicin, epicholesterol and selected phospholipids, favor the inward-facing state, suggesting uptake or allosteric stabilization. Thus, conserved proton-coupling elements can power opposing transport modes, revealing the mechanistic versatility of the Spns fold and its therapeutic potential.
Reza Dastvan, Samantha Gies, Kevin L. Jagessar et al.· Research Square· 0 citations
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