The Molecular Basis of N-End Rule Recognition

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Molecular cell (Impact Factor: 14.02). 12/2008; 32(3):406-14. DOI: 10.1016/j.molcel.2008.08.032
Source: PubMed


The N-end rule targets specific proteins for destruction in prokaryotes and eukaryotes. Here, we report a crystal structure of a bacterial N-end rule adaptor, ClpS, bound to a peptide mimic of an N-end rule substrate. This structure, which was solved at a resolution of 1.15 A, reveals specific recognition of the peptide alpha-amino group via hydrogen bonding and shows that the peptide's N-terminal tyrosine side chain is buried in a deep hydrophobic cleft that pre-exists on the surface of ClpS. The adaptor side chains that contact the peptide's N-terminal residue are highly conserved in orthologs and in E3 ubiquitin ligases that mediate eukaryotic N-end rule recognition. We show that mutation of critical ClpS contact residues abrogates substrate delivery to and degradation by the AAA+ protease ClpAP, demonstrate that modification of the hydrophobic pocket results in altered N-end rule specificity, and discuss functional implications for the mechanism of substrate delivery.

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    • "It was previously shown that the addition of a chloride moiety to unmodified amphetamine substantially increased its inhibitory efficiency43. Although the chloride atom substitution of PCA may facilitate the interaction between PCA and the active sites by allowing water molecules to occupy the space and mediate hydrogen bonds as seen in the UBR box and ClpS domain1944, the structural basis of inhibitory effects of PCA is to be determined. The para-positioned chlorine of PCA may function as an essential pharmacophore in the inhibition, which can be a target for further structure-activity relationship study. "
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    ABSTRACT: In the arginylation branch of the N-end rule pathway, unacetylated N-terminal destabilizing residues function as essential determinants of protein degradation signals (N-degron). Here, we show that a neurostimulant, para-chloroamphetamine (PCA), specifically inhibits the Arg/N-end rule pathway, delaying the degradation of its artificial and physiological substrates, including regulators of G protein signaling 4 (RGS4), in vitro and in cultured cells. In silico computational analysis indicated that PCA strongly interacts with both UBR box and ClpS box, which bind to type 1 and type 2 N-degrons, respectively. Moreover, intraperitoneal injection of PCA significantly stabilized endogenous RGS4 proteins in the whole mouse brain and, particularly, in the frontal cortex and hippocampus. Consistent with the role of RGS4 in G protein signaling, treatment with PCA impaired the activations of GPCR downstream effectors in N2A cells, phenocopying ATE1-null mutants. In addition, levels of pathological C-terminal fragments of TDP43 bearing N-degrons (Arg208-TDP25) were significantly elevated in the presence of PCA. Thus, our study identifies PCA as a potential tool to understand and modulate various pathological processes regulated by the Arg/N-end rule pathway, including neurodegenerative processes in FTLD-U and ALS.
    Scientific Reports 09/2014; 4:6344. DOI:10.1038/srep06344 · 5.58 Impact Factor
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    • "The current data suggest a model whereby the degradation of a bacterial N-end rule substrate by ClpAPS is a highly coordinated process. In the first step, the target protein containing a type 2 destabilizing residue at its N-terminus is bound by the hydrophobic pocket on ClpS, which involves a network of specific interactions with the α-amino group and the side chain of the Nterminal residue and the first peptide bond [22] [23] [46]. Upon recognition of the substrate by ClpS, the ClpS-substrate complex is delivered to ClpA in two steps; initially through docking to the Nterminal domain of ClpA, which results in activation of ClpA by the Nterminal tail of ClpS, to receive the N-end rule substrate [70] [71] [72] [73]. "
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    ABSTRACT: Intracellular proteolysis is a tightly regulated process responsible for the targeted removal of unwanted or damaged proteins. The non-lysosomal removal of these proteins is performed by processive enzymes, which belong to the AAA+superfamily, such as the 26S proteasome and Clp proteases. One important protein degradation pathway, that is common to both prokaryotes and eukaryotes, is the N-end rule. In this pathway, proteins bearing a destabilizing amino acid residue at their N-terminus are degraded either by the ClpAP protease in bacteria, such as Escherichia coli or by the ubiquitin proteasome system in the eukaryotic cytoplasm. A suite of enzymes and other molecular components are also required for the successful generation, recognition and delivery of N-end rule substrates to their cognate proteases. In this review we examine the similarities and differences in the N-end rule pathway of bacterial and eukaryotic systems, focusing on the molecular determinants of this pathway.
    Biochimica et Biophysica Acta 07/2011; 1823(1):83-91. DOI:10.1016/j.bbamcr.2011.07.002 · 4.66 Impact Factor
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    • "In addition to the L / F transferase , the Clp protease components ClpA / P and ClpS play a crucial role in the bacterial N - end rule pathway ( Tobias et al . , 1991 ; Erbse et al . , 2006 ; Wang et al . , 2008 ; Schmidt et al . , 2009 ) . It seems reasonable to speculate that the chloroplast homologue of the bacterial Clp protease could be also involved in degrad - ing plastid proteins with unstable N - terminal sequences . However , although plastids possess numerous isoforms of subunits of the Clp protease core complex , the ClpS adapter pr"
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    ABSTRACT: Although chloroplast protein stability has long been recognised as a major level of post-translational regulation in photosynthesis and gene expression, the factors determining protein stability in plastids are largely unknown. Here, we have identified stability determinants in vivo by producing plants with transgenic chloroplasts that express a reporter protein whose N- and C-termini were systematically modified. We found that major stability determinants are located in the N-terminus. Moreover, testing of all 20 amino acids in the position after the initiator methionine revealed strong differences in protein stability and indicated an important role of the penultimate N-terminal amino acid residue in determining the protein half life. We propose that the stability of plastid proteins is largely determined by three factors: (i) the action of methionine aminopeptidase (the enzyme that removes the initiator methionine and exposes the penultimate N-terminal amino acid residue), (ii) an N-end rule-like protein degradation pathway, and (iii) additional sequence determinants in the N-terminal region.
    The Plant Journal 08/2010; 63(4):636-50. DOI:10.1111/j.1365-313X.2010.04268.x · 5.97 Impact Factor
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