The Molecular Basis of N-End Rule Recognition

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Molecular cell (Impact Factor: 14.46). 12/2008; 32(3):406-14. DOI: 10.1016/j.molcel.2008.08.032
Source: PubMed

ABSTRACT 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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    • "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 · 6.82 Impact Factor
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    • "Consistent with the notion that the volume of the pocket is an important determinant of substrate binding, two hydrophobic residues (M40 and M62) that line the cavity modulate the volume of the cavity and, hence, its specificity. Substitution of one residue (M40A) was sufficient to expand the binding repertoire of ClpS to include Ile, albeit weakly, while replacement of both residues (M40A/M62A) improved the recognition, and hence degradation of a substrate bearing an N-terminal Trp (Wang et al., 2008a; Schuenemann et al., 2009; S. Kralik, K. Zeth and D.A. Dougan, unpublished). Nevertheless, despite this structural plasticity, the binding pocket is unable to accommodate the side-chain of Met or b-branched amino acids such as Ile and Val. "
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    ABSTRACT: The N-end rule pathway is a highly conserved process that operates in many different organisms. It relates the metabolic stability of a protein to its N-terminal amino acid. Consequently, amino acids are described as either 'stabilizing' or 'destabilizing'. Destabilizing residues are organized into three hierarchical levels: primary, secondary, and in eukaryotes - tertiary. Secondary and tertiary destabilizing residues act as signals for the post-translational modification of the target protein, ultimately resulting in the attachment of a primary destabilizing residue to the N-terminus of the protein. Regardless of their origin, proteins containing N-terminal primary destabilizing residues are recognized by a key component of the pathway. In prokaryotes, the recognition component is a specialized adaptor protein, known as ClpS, which delivers target proteins directly to the ClpAP protease for degradation. In contrast, eukaryotes use a family of E3 ligases, known as UBRs, to recognize and ubiquitylate their substrates resulting in their turnover by the 26S proteasome. While the physiological role of the N-end rule pathway is largely understood in eukaryotes, progress on the bacterial pathway has been slow. However, new interest in this area of research has invigorated several recent advances, unlocking some of the secrets of this unique proteolytic pathway in prokaryotes.
    Molecular Microbiology 03/2010; 76(3):545-58. DOI:10.1111/j.1365-2958.2010.07120.x · 5.03 Impact Factor
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