Argonaute proteins (Agos) from thermophilic archaea are involved in several important processes, such as host defense and DNA replication. The catalytic mechanism of Ago from different microbes with great diversity and genome editing potential is attracting increasing attention. Here, we describe an Argonaute from hyperthermophilic Ferroglobus placidus (FpAgo), with a typical DNA-guided DNA endonuclease activity but adopted with only a short guide 15–20 nt length rather than a broad guide selectivity for reported Agos. FpAgo performed the precise cleavage of phosphodiester bonds between 10 and 11 nt on the target strand (counting from the guide strand) guided strictly by 5′-phosphorylated DNA at temperatures ranging from 75 to 99°C. The cleavage activity was regulated by the divalent cations Mn2+, Mg2+, Co2+, and Ni2+. In addition, FpAgo possesses guide/target mismatch tolerance in the seed region but is sensitive to mismatches in the 3′-guide region. Notably, the EMSA assay revealed that the FpAgo-guide-target ternary complex exhibited a stronger binding affinity for short 15 and 16 nt guide DNAs than longer guides. Moreover, we performed structural modeling analyses that implied the unique PAZ domain of FpAgo for 3′-guide recognition and binding to affect guide length specificity. This study broadens our understanding of thermophilic Agos and paves the way for their use in DNA manipulation.
Caldicellulosiruptor bescii is the most thermophilic cellulolytic species of organisms known to date. In our previous study, GH10 xylanase CbXyn10B from C. bescii displayed outstanding hydrolytic activity toward various xylans at high temperatures. To understand the structural basis for this protein's catalysis and thermostability, we solved the crystal structures of CbXyn10B and its complexes with xylooligosaccharides. These structural models were used to guide comparison with its mesophilic counterpart PbXyn10B. A distinctive structural feature is that thermophilic CbXyn10B presents a relatively stable interaction between the extended loops L7 and L8 in the catalytic cleft by an extensive hydrogen bonding network, which is mediated by Lys, Arg and three well-ordered water molecules. Moreover, a unique aromatic cluster consisting of Try, Phe, Phe, and Phe may enhance the interaction between the N- and C- terminus. Targeted mutagenesis demonstrated that these interactions substantially contribute to enzyme stabilization, as indicated by a considerable decrease in the melting temperature (T) of CbXyn10B by substituting critical residues with Ala. Therefore, it was shown that not only the aromatic interaction connecting protein termini but also the extensive hydrogen bonding network formed between surface loops could restrict the local structural flexibility and contribute significantly to the overall stability of enzymes. Furthermore, the xylooligosaccharides were found to tightly bind to the glycone subsites of xylanase, indicating higher affinities at these subsites and reflecting its substrate binding preference. Our results suggest that CbXyn10B is stabilized with distinct rigidity at the catalytic cleft as well as the terminal regions, which provides insights into the evolutionary strategy for accommodating the functional needs of GH10 enzymes to high temperature.
Mapping NAD+ dynamics in live cells and human
is essential
for translating NAD+ interventions into effective therapies.
Yet, genetically encoded NAD+ sensors with better specificity
and pH resistance are still needed for the cost-effective monitoring
of NAD+ in both subcellular compartments and clinical samples.
Here, we introduce multicolor, resonance energy transfer-based NAD+ sensors covering nano- to millimolar concentration ranges
for clinical NAD+ measurement and subcellular NAD+ visualization. The sensors captured the blood NAD+ increase
induced by NMN supplementation and revealed the distinct subcellular
effects of NAD+ precursors and modulators. The sensors
then enabled high-throughput screenings for mitochondrial and nuclear
NAD+ modulators and identified α-GPC, a cognition-related
metabolite that induces NAD+ redistribution from mitochondria
to the nucleus relative to the total adenine nucleotides, which was
further confirmed by NAD+ FRET microscopy.
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