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    • ApexPrep DNA Plasmid Miniprep Column Only br RING type domai

    2020-07-31


    RING-type domain structure RING structure is conformed as a consequence of a cross-braced arrangement of eight Zn coordinating residues, generally Cys and His, with conserved spacing between these residues (Fig. 2B and C). Canonical RINGs have either one or two His in the linear arrangement of coordinating residues, denoted C3H2C3 or C3HC4, however other variations exist. The PHD/LAP finger found in the transcription factor NF-X1 and the MARCH family of membrane-bound E3s is defined by its C4HC3 consensus. RINGs having a C8 configuration (CNOT4) or an Asp residue in the final position (e.g. Rbx1 and TRAF6) have been shown to have ligase activity [74], [75], [76]. Thus, it has become apparent that categorizing RINGs by the linear arrangement of coordinating residues has little to do with functional properties of the domain. Nevertheless, context does matter, as swapping Zn liganding residues in a C3H2C3 RING to create a C3HC4 configuration resulted in loss of activity for AO7 (RNF25), one of the first RING E3s studied [50]. NF-X1 contains a sequence in which both a RING and a PHD/LAP motif are recognizable, but only the PHD/LAP consensus is functional [77]. Unlike RING domain E3s, U-box proteins do not coordinate Zn but adopt a RING-like tertiary structure for binding E2, stabilized by non-covalent interactions among core ApexPrep DNA Plasmid Miniprep Column Only [16]. Additionally, some pathogenic bacteria have evolved proteins that show no sequence homology to eukaryotic RING or U-box domains, yet fold into highly similar structures and display robust ubiquitin ligase activity [78], [79]. Crystallographic and NMR-based analyses have revealed that RINGs and U-boxes have a common mode of interaction with E2s (Fig. 2A). The key structural elements are two loop-like regions, which, in the case of RINGs, coordinate Zn. The loops surround a shallow groove formed by the central α-helix. Together these elements serve as a platform for interactions with the UBC domain of E2s (Fig. 2A). The E2 surface that interacts with the RING domain overlaps with the region that interacts with E1, leading to the notion that dissociation of E2s from RINGs is required for an E2 to be ‘reloaded’ with ubiquitin by E1 [80], [81], [82]. A characteristic of RING:E2 interactions is that they are generally of low affinity, typically with Kd values in the high micromolar range. Thus, even though a RING domain may function robustly with an E2, assessing physical interactions between these proteins using standard ‘pulldown’ approaches is often not fruitful. Exceptions to this feature include E3s such as gp78 [83], Rad18 [84], and AO7 [50] (S. Li, Y. Liang, X. Ji, & A.M.W., unpublished observations), which contain regions outside the RING motif that bind E2s through distinct interfaces, resulting in high affinity interactions (see below). RING:E2 interactions typically involve conserved, bulky hydrophobic side chains. Mutation of these side chains mitigates E2 binding and causes decreased levels of ubiquitination activity in vitro, as demonstrated for c-Cbl Trp408Ala [85], CNOT4 Leu16Ala [74], and BRCA1 Ile26Ala [86]. However, because a given RING can function with a cohort of E2s with varying binding affinities [86], [87], mutation of such RING domain:E2 residues may yield unexpected results. For example, residual activity is observed in vitro for the BRCA1 Ile26Ala mutant with select E2 pairings (J.N.P., D.M. Wenzel, & R.E.K., unpublished observations), and the analogous mutation in other RING E3s does not consistently eliminate activity (J. Callis, UC Davis, personal communications). Thus, the relationship between E2 binding and activity remains to be fully characterized and will require mutants where, in the context of a correctly folded RING (i.e. retaining its Zn-coordinating residues), E2 binding is abolished. Conversely, in vivo analysis of RING function demands a truly ‘ligase-dead’ mutant that retains E2~Ub binding. Identification of such mutants awaits a more thorough definition of RING catalytic function.