The Proteomics of N-terminal Methionine Cleavage

Frottin, Frédéric; Martinez, Aude; Peynot, Philippe; Mitra, Sanghamitra; Holz, Richard C.; Giglione, Carmela; Meinnel, Thierry

doi:10.1074/mcp.m600225-mcp200

Cited by 345 publications

(387 citation statements)

References 67 publications

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“…The molecular masses measured by MALDI-TOF for NiV P XD (6740 Ϯ 3 Da) and for HeV P XD (6737 Ϯ 3 Da) are consistent with the values expected for full-length proteins in which the initial methionine is either conserved (NiV) or cleaved off (HeV). These findings are in agreement with previous studies showing that in proteins expressed in E. coli the initial methionine is generally cleaved off when followed by small residues such as glycine or alanine (81).…”

Section: Purification Of Henipavirus N Proteins and Assessment Of Thesupporting

confidence: 83%

Characterization of the Interactions between the Nucleoprotein and the Phosphoprotein of Henipavirus

Habchi

Blangy

Mamelli

et al. 2011

Journal of Biological Chemistry

165

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The Henipavirus genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). In a previous study, we reported that in henipaviruses, the N-terminal domain of the phosphoprotein and the C-terminal domain of the nucleoprotein (N TAIL ) are both intrinsically disordered. Here we show that Henipavirus N TAIL domains are also disordered in the context of full-length nucleoproteins. We also report the cloning, purification, and characterization of the C-terminal X domains (P XD ) of Henipavirus phosphoproteins. Using isothermal titration calorimetry, we show that N TAIL and P XD form a 1:1 stoichiometric complex that is stable under NaCl concentrations as high as 1 M and has a K D in the M range. Using far-UV circular dichroism and nuclear magnetic resonance, we show that P XD triggers an increase in the ␣-helical content of N TAIL . Using fluorescence spectroscopy, we show that P XD has no impact on the chemical environment of a Trp residue introduced at position 527 of the Henipavirus N TAIL domain, thus arguing for the lack of stable contacts between the C termini of N TAIL and P XD . Finally, we present a tentative structural model of the N TAIL -P XD interaction in which a short, order-prone region of N TAIL (␣-MoRE; amino acids 473-493) adopts an ␣-helical conformation and is embedded between helices ␣2 and ␣3 of P XD , leading to a relatively small interface dominated by hydrophobic contacts. The present results provide the first detailed experimental characterization of the N-P interaction in henipaviruses and designate the N TAIL -P XD interaction as a valuable target for rational antiviral approaches.

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Section: Purification Of Henipavirus N Proteins and Assessment Of Thesupporting

confidence: 83%

Characterization of the Interactions between the Nucleoprotein and the Phosphoprotein of Henipavirus

Habchi

Blangy

Mamelli

et al. 2011

Journal of Biological Chemistry

165

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“…Nevertheless, proteins with N-terminal Met are not recognized as N-end rule substrates (9,13). This selectivity is biologically important, because the majority of bacterial proteins have an N-terminal Met (19,20) We considered the possibility that Met might be sterically Values in parentheses are for the highest resolution bin. Rsym ϭ ⌺h⌺j ԽIj(h) Ϫ ͗I(h)͘Խ / ⌺h⌺j ͗I(h)͘, where Ij(h) is the jth reflection of index h and ͗I(h)͘ is the average intensity of all observations of I(h).…”

Section: Resultsmentioning

confidence: 99%

“…Indeed based on predictions and direct measurements, between 2% and 3% of proteins in the E. coli proteome are thought to begin with Val, because methionine aminopeptidase removes the initiator Met from proteins that are synthesized with Met-Val at the N terminus (19,22,23). Wang et al (17) demonstrated that the Met 53 side chain in ClpS plays a major role in excluding Val and Ile, because the restriction against these ␤-branched residues was relieved when the ''gatekeeper'' Met was replaced by Ala.…”

Section: Discussionmentioning

confidence: 99%

Molecular basis of substrate selection by the N-end rule adaptor protein ClpS

Roman-Hernandez¹,

Grant²,

Sauer³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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The N-end rule is a conserved degradation pathway that relates the stability of a protein to its N-terminal amino acid. Here, we present crystal structures of ClpS, the bacterial N-end rule adaptor, alone and engaged with peptides containing N-terminal phenylalanine, leucine, and tryptophan. These structures, together with a previous structure of ClpS bound to an N-terminal tyrosine, illustrate the molecular basis of recognition of the complete set of primary N-end rule amino acids. In each case, the ␣-amino group and side chain of the N-terminal residue are the major determinants of recognition. The binding pocket for the N-end residue is preformed in the free adaptor, and only small adjustments are needed to accommodate N-end rule residues having substantially different sizes and shapes. M53A ClpS is known to mediate degradation of an expanded repertoire of substrates, including those with N-terminal valine or isoleucine. A structure of Met53A ClpS engaged with an N-end rule tryptophan reveals an essentially wild-type mechanism of recognition, indicating that the Met 53 side chain directly enforces specificity by clashing with and excluding ␤-branched side chains. Finally, experimental and structural data suggest mechanisms that make proteins with N-terminal methionine bind very poorly to ClpS, explaining why these high-abundance proteins are not degraded via the N-end rule pathway in the cell.AAAϩ ATPase adaptor ͉ clpAP protease ͉ degradation tag ͉ degron

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“…Considering the 177 proteins that retain the initial Met (no NME or partial NME), a small set (13 hits with no-NME and 5 with partial NME), which was wrongly predicted, is related to proteins with Thr (10 of 18), Val (5 of 18), Ala (2 of 18), and Ser (1 of 18) at position 2. Indeed, Thr and Val residues are known to have a negative influence on NME efficiency and other amino acids beyond the penultimate residue strongly influence the cleavage (Thr, Pro, Glu, Asp…) making prediction sometimes difficult (50). In such cases, partial cleavage is expected as observed for proteins P12236, Q9GZS3, or Q9NPJ3.…”

Section: N-terminalmentioning

confidence: 99%