MUTAGENESIS OF ASP-326; GLU-422; ARG-490 AND ASP-510
STRUCTURE BY ELECTRON MICROSCOPY (3.00 ANGSTROMS) IN COMPLEX WITH GUIDE RNA; TARGET DNA AND ZINC
CRISPR-associated endodeoxyribonuclease Cas12f1
CRISPR-associated endodeoxyribonuclease Cas14a1
CRISPR (clustered regularly interspaced short palindromic repeat), is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain sequences complementary to antecedent mobile elements and target invading nucleic acids (Probable). CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA), which requires a trans-encoded small RNA (tracrRNA), but not this protein (in vitro) (Probable). Upon expression in E.coli of this protein, a mini CRISPR array and the probable tracrRNA, the protein associates with both RNAs (PubMed:30337455). The mini system is not active in E.coli against phiX174 phage, nor is it active in protection against transformation by foreign plasmids (PubMed:30337455, PubMed:32246713). In vitro the purified protein-tracrRNA-crRNA complex cleaves ssDNA complementary to the crRNA; target cleavage requires both tracrRNA and crRNA, but not a protospacer adjacent motif (PAM). The tracrRNA-crRNA can be replaced by a single guide RNA (sgRNA). 2-nucleotide mismatches in the middle of the crRNA:DNA heteroduplex decrease cleavage. Cleavage occurs just downstream of the heteroduplex (PubMed:30337455). Activation of this protein results in non-specific ssDNA degradation in vitro (PubMed:30337455, PubMed:32246713). In vitro and in E.coli (coexpressed with sgRNA) has dsDNA endonuclease activity, recognizing the 5' PAM sequence TTTR; both sgRNA and a PAM are required for activity. Cleaves the target strand 24 and the nontarget strand 22 bases upstream of the PAM (respectively), resulting in 5' overhangs (PubMed:32246713, PubMed:33333018). The 2 monomers interact differently with the sgRNA and target DNA. Mutagenesis of a dimeric construct shows that one of the RuvC monomers probably cleaves both DNA strands (PubMed:33333018).
Mg(2+) is required for dsDNA cleavage (PubMed:32246713). Mg(2+) and Mn(2+) support ssDNA cleavage equally (PubMed:30337455).
Homodimeric Cas12f in complex with sgRNA and target DNA binds only 3 Zn(2+) ions.
Target ssDNA cleavage is inhibited by EDTA (PubMed:30337455). Activity is maximal with 5-50 mM NaCl, is less efficient at higher NaCl concentrations (PubMed:32246713, PubMed:33333018).
Optimum temperature for dsDNA cleavage is 46 degrees Celsius and occurs between 37-52 degrees Celsius, no cleavage at 55 degrees Celsius (PubMed:32246713). Optimum temperature for ssDNA cleavage is 45-55 degrees Celsius (PubMed:30337455).
An asymmetric homodimer. Guide RNA is probably required for dimerization.
Has an asymmetric bilobed structure, with a recognition (REC) lobe and nuclease (NUC) lobe; the sgRNA:target DNA is bound between the 2 lobes. The REC lobe (residues 1-312) is formed by the WED, ZF and REC domains, while the NUC lobe (residues 321-529) is formed by the RuvC and TNB domains. Has a split RuvC-like domain with the nuclease active sites.
The requirement for a perfect match in the middle of the heteroduplex, joined with its lack of a PAM, allows its use in detection of single-nucleotide polymorphisms (a DETECTR assay). The tracrRNA-crRNA can be replaced by a single guide RNA (sgRNA) (PubMed:30337455). Its inability to protect against plasmid transformation suggests there are elements missing in E.coli which prevents its use in more classic genome editing (PubMed:32246713).
Belongs to the CRISPR-associated endonuclease Cas12f family.
Loss of cleavage of target dsDNA cleavage, binds sgRNA; when associated with R-178.
Almost complete loss of target dsDNA cleavage.
About 40% cleavage of target dsDNA, loss of cleavage, binds sgRNA; when associated with 118-R--R-126.
Nearly wild-type cleavage of target dsDNA.
No cleavage of target ssDNA. No cleavage of supercoiled target dsDNA.
X-RAY CRYSTALLOGRAPHY (2.3 ANGSTROMS) OF 25-535
Exhibits fusogenic activity (PubMed:35794124). Mediates cell-cell fusion in mammalian cells when present in both cells (bilateral fusion) (PubMed:35794124).
Monomer in solution, crystallizes as a trimer in high salt (2.5 M NaCl, 0.2 M CaCl(2)). The trimer is stabilized by interdomain contacts and numerous Ca(2+) and Na(+) ions.
The extracellular N-terminus has 4 domains; the first 3 are structurally similar to fusogens from plants, C.elegans and viruses, while the fourth domain is unique to archaea. Domains I and II are discontinuous, domain IV cannot be replaced. The protein can be anchored to the membrane by either a different transmembrane helix or by a GPI-anchored construct and still directs membrane fusion. The fusion loop in domain II is stabilized by a Ca(2+) ion so that it protrudes from the molecule.
This entry represents a metagenomic sequence isolated from an uncultured archaeon from a hypersaline environment.
Belongs to the HAP2/GCS1 family. Fusexin 1 subfamily.
Fusion loop, required for fusogenic activity, not required for membrane surface localization
Domain IV, required for fusogenic activity
Loss of fusogenic activity, still found on cell surface (in mammalian cells).
X-RAY CRYSTALLOGRAPHY (1.53 ANGSTROMS) IN COMPLEX WITH NAD AND MAGNESIUM IONS
KETOL-ACID REDUCTOISOMERASE, MITOCHONDRIAL
6-phosphogluconate dehydrogenase C-terminal domain-like
Alpha-keto-beta-hydroxylacyl reductoisomerase
Involved in the biosynthesis of branched-chain amino acids (BCAA). Catalyzes an alkyl-migration followed by a ketol-acid reduction of (S)-2-acetolactate (S2AL) to yield (R)-2,3-dihydroxy-isovalerate. In the isomerase reaction, S2AL is rearranged via a Mg-dependent methyl migration to produce 3-hydroxy-3-methyl-2-ketobutyrate (HMKB). In the reductase reaction, this 2-ketoacid undergoes a metal-dependent reduction by NADH to yield (R)-2,3-dihydroxy-isovalerate.
Belongs to the ketol-acid reductoisomerase family.