Library Screening, In Vivo Confirmation, and Structural and Bioinformatic Analysis of Pentapeptide Sequences as Substrates for Protein Farnesyltransferase

Schey, Garrett L.; Hildebrandt, Emily R.; Wang, You; Diwan, Safwan; Passetti, Holly A.; Potts, Gavin W.; Sprague-Getsy, Andrea M.; Leoni, Ethan R.; Kuebler, Taylor S.; Sham, Yuk Y.; Hougland, James L.; Beese, Lorena S.; Schmidt, Walter K.; Distefano, Mark D.

doi:10.3390/ijms25105324

Title: Library Screening, In Vivo Confirmation, and Structural and Bioinformatic Analysis of Pentapeptide Sequences as Substrates for Protein Farnesyltransferase

Journal Article · 13 May 2024 · International Journal of Molecular Sciences (Online)

DOI: https://doi.org/10.3390/ijms25105324 · OSTI ID:2470081

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^[2]; Wang, You ^[3];

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^[1]; Potts, Gavin W. ^[1]; Sprague-Getsy, Andrea M. ^[4];

^[2]; Kuebler, Taylor S. ^[1];

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Univ. of Minnesota, Minneapolis, MN (United States)
Univ. of Georgia, Athens, GA (United States)
Duke Univ. School of Medicine, Durham, NC (United States)
Syracuse Univ., NY (United States)

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization–Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES)

Grant/Contract Number:: AC02-06CH11357; W-31109-ENG-38

OSTI ID:: 2470081

Journal Information:: International Journal of Molecular Sciences (Online), Journal Name: International Journal of Molecular Sciences (Online) Journal Issue: 10 Vol. 25; ISSN 1422-0067

Publisher:: MDPICopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
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peptide libraries
protein prenylation

Title: Library Screening, In Vivo Confirmation, and Structural and Bioinformatic Analysis of Pentapeptide Sequences as Substrates for Protein Farnesyltransferase

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