HD domain

Abstract: Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase+/nuclease− and ATPase−/nuclease+) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade–crRNA complex targeting foreign DNA.

Abstract: The synthesis of extracellular enzymes and extracellular polysaccharide (EPS) in Xanthomonas campestris pv. campestris (Xcc) is regulated by a cluster of genes called rpf (for regulation of pathogenicity factors). Two of the genes, rpfF and rpfB, have previously been implicated in the synthesis of a diffusible regulatory molecule, DSF. Here, we describe a screen of transposon insertion mutants of Xcc that identified two DSF-overproducing strains. In each mutant, the gene disrupted is rpfC, which encodes a hybrid two-component regulatory protein in which the sensor and regulator domains are fused and which contains an additional C-terminal phosphorelay (HPt) domain. We show that rpfC is in an operon with rpfH and rpfG. The predicted protein RpfG has a regulatory input domain attached to a specialized version of an HD domain, previously suggested to function in signal transduction. The predicted protein RpfH is structurally related to the sensory input domain of RpfC. We show that RpfC and RpfG act positively to regulate the synthesis of extracellular enzymes and EPS, but that RpfC acts negatively to regulate the synthesis of DSF. We propose that RpfGHC is a signal transduction system that couples the synthesis of pathogenicity factors to sensing of environmental signals that may include DSF itself.

Abstract: The NOTCH1 receptor is cleaved within its extracellular domain by furin during its maturation, yielding two subunits that are held together noncovalently by a juxtamembrane heterodimerization (HD) domain. Normal NOTCH1 signaling is initiated by the binding of ligand to the extracellular subunit, which renders the transmembrane subunit susceptible to two successive cleavages within and C terminal to the heterodimerization domain, catalyzed by metalloproteases and gamma-secretase, respectively. Because mutations in the heterodimerization domain of NOTCH1 occur frequently in human T-cell acute lymphoblastic leukemia (T-ALL), we assessed the effect of 16 putative tumor-associated mutations on Notch1 signaling and HD domain stability. We show here that 15 of the 16 mutations activate canonical NOTCH1 signaling. Increases in signaling occur in a ligand-independent fashion, require gamma-secretase activity, and correlate with an increased susceptibility to cleavage by metalloproteases. The activating mutations cause soluble NOTCH1 heterodimers to dissociate more readily, either under native conditions (n = 3) or in the presence of urea (n = 11). One mutation, an insertion of 14 residues immediately N terminal to the metalloprotease cleavage site, increases metalloprotease sensitivity more than all others, despite a negligible effect on heterodimer stability by comparison, suggesting that the insertion may expose the S2 site by repositioning it relative to protective NOTCH1 ectodomain residues. Together, these studies show that leukemia-associated HD domain mutations render NOTCH1 sensitive to ligand-independent proteolytic activation through two distinct mechanisms.