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Context-dependent substrate recognition by protein farnesyltransferase.

ABSTRACT: Prenylation is a posttranslational modification whereby C-terminal lipidation leads to protein localization to membranes. A C-terminal "Ca(1)a(2)X" sequence has been proposed as the recognition motif for two prenylation enzymes, protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I. To define the parameters involved in recognition of the a(2) residue, we performed structure-activity analysis which indicates that FTase discriminates between peptide substrates based on both the hydrophobicity and steric volume of the side chain at the a(2) position. For nonpolar side chains, the dependence of the reactivity on side chain volume at this position forms a pyramidal pattern with a maximal activity near the steric volume of valine. This discrimination occurs at a step in the kinetic mechanism that is at or before the farnesylation step. Furthermore, a(2) selectivity is also affected by the identity of the adjacent X residue, leading to context-dependent substrate recognition. Context-dependent a(2) selectivity suggests that FTase recognizes the sequence downstream of the conserved cysteine as a set of two or three cooperative, interconnected recognition elements as opposed to three independent amino acids. These findings expand the pool of proposed FTase substrates in cells. A better understanding of the molecular recognition of substrates performed by FTase will aid in both designing new FTase inhibitors as therapeutic agents and characterizing proteins involved in prenylation-dependent cellular pathways.

SUBMITTER: Hougland JL

PROVIDER: S-EPMC2765569 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Context-dependent substrate recognition by protein farnesyltransferase.

Hougland James L JL Lamphear Corissa L CL Scott Sarah A SA Gibbs Richard A RA Fierke Carol A CA

Biochemistry 20090301 8

Prenylation is a posttranslational modification whereby C-terminal lipidation leads to protein localization to membranes. A C-terminal "Ca(1)a(2)X" sequence has been proposed as the recognition motif for two prenylation enzymes, protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I. To define the parameters involved in recognition of the a(2) residue, we performed structure-activity analysis which indicates that FTase discriminates between peptide substrates based on b ...[more]

PMID: 19199818

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Project description:Prenylation is a posttranslational modification essential for the proper localization and function of many proteins. Farnesylation, the attachment of a 15-carbon farnesyl group near the C-terminus of protein substrates, is catalyzed by protein farnesyltransferase (FTase). Farnesylation has received significant interest as a target for pharmaceutical development, and farnesyltransferase inhibitors are in clinical trials as cancer therapeutics. However, as the total complement of prenylated proteins is unknown, the FTase substrates responsible for farnesyltransferase inhibitor efficacy are not yet understood. Identifying novel prenylated proteins within the human proteome constitutes an important step towards understanding prenylation-dependent cellular processes. Based on sequence preferences for FTase derived from analysis of known farnesylated proteins, we selected and screened a library of small peptides representing the C-termini of 213 human proteins for activity with FTase. We identified 77 novel FTase substrates that exhibit multiple-turnover (MTO) reactivity within this library; our library also contained 85 peptides that can be farnesylated by FTase only under single-turnover (STO) conditions. Based on these results, a second library was designed that yielded an additional 29 novel MTO FTase substrates and 45 STO substrates. The two classes of substrates exhibit different specificity requirements. Efficient MTO reactivity correlates with the presence of a nonpolar amino acid at the a(2) position and a Phe, Met, or Gln at the terminal X residue, consistent with the proposed Ca(1)a(2)X sequence model. In contrast, the sequences of the STO substrates vary significantly more at both the a(2) and the X residues and are not well described by current farnesylation algorithms. These results improve the definition of prenyltransferase substrate specificity, test the efficacy of substrate algorithms, and provide valuable information about therapeutic targets. Finally, these data illuminate the potential for in vivo regulation of prenylation through modulation of STO versus MTO peptide reactivity with FTase.

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Context-dependent substrate recognition by protein farnesyltransferase.

Publications

Context-dependent substrate recognition by protein farnesyltransferase.

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