Targeted reengineering of protein geranylgeranyltransferase type i selectivity functionally implicates active-site residues in protein-substrate recognition

Soumyashree A. Gangopadhyay, Erica L. Losito, James L Hougland

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Posttranslational modifications are vital for the function of many proteins. Prenylation is one such modification, wherein protein geranylgeranyltransferase type I (GGTase-I) or protein farnesyltransferase (FTase) modify proteins by attaching a 20- or 15-carbon isoprenoid group, respectively, to a cysteine residue near the C-terminus of a target protein. These enzymes require a C-terminal Ca1a2X sequence on their substrates, with the a1, a2, and X residues serving as substrate-recognition elements for FTase and/or GGTase-I. While crystallographic structures of rat GGTase-I show a tightly packed and hydrophobic a2 residue binding pocket, consistent with a preference for moderately sized a2 residues in GGTase-I substrates, the functional impact of enzyme-substrate contacts within this active site remains to be determined. Using site-directed mutagenesis and peptide substrate structure-activity studies, we have identified specific active-site residues within rat GGTase-I involved in substrate recognition and developed novel GGTase-I variants with expanded/altered substrate selectivity. The ability to drastically alter GGTase-I selectivity mirrors similar behavior observed in FTase but employs mutation of a distinct set of structurally homologous active-site residues. Our work demonstrates that tunable selectivity may be a general phenomenon among multispecific enzymes involved in posttranslational modification and raises the possibility of variable substrate selectivity among GGTase-I orthologues from different organisms. Furthermore, the GGTase-I variants developed herein can serve as tools for studying GGTase-I substrate selectivity and the effects of prenylation pathway modifications on specific proteins.

Original languageEnglish (US)
Pages (from-to)434-446
Number of pages13
JournalBiochemistry
Volume53
Issue number2
DOIs
StatePublished - Jan 21 2014

Fingerprint

Reengineering
Catalytic Domain
Substrates
Proteins
Farnesyltranstransferase
Prenylation
Post Translational Protein Processing
geranylgeranyltransferase type-I
Rats
Enzymes
Mutagenesis
Aptitude
Terpenes
Site-Directed Mutagenesis
Cysteine
Carbon

ASJC Scopus subject areas

  • Biochemistry

Cite this

Targeted reengineering of protein geranylgeranyltransferase type i selectivity functionally implicates active-site residues in protein-substrate recognition. / Gangopadhyay, Soumyashree A.; Losito, Erica L.; Hougland, James L.

In: Biochemistry, Vol. 53, No. 2, 21.01.2014, p. 434-446.

Research output: Contribution to journalArticle

@article{f3780dc6094f4085bc50e2a1daa9472d,
title = "Targeted reengineering of protein geranylgeranyltransferase type i selectivity functionally implicates active-site residues in protein-substrate recognition",
abstract = "Posttranslational modifications are vital for the function of many proteins. Prenylation is one such modification, wherein protein geranylgeranyltransferase type I (GGTase-I) or protein farnesyltransferase (FTase) modify proteins by attaching a 20- or 15-carbon isoprenoid group, respectively, to a cysteine residue near the C-terminus of a target protein. These enzymes require a C-terminal Ca1a2X sequence on their substrates, with the a1, a2, and X residues serving as substrate-recognition elements for FTase and/or GGTase-I. While crystallographic structures of rat GGTase-I show a tightly packed and hydrophobic a2 residue binding pocket, consistent with a preference for moderately sized a2 residues in GGTase-I substrates, the functional impact of enzyme-substrate contacts within this active site remains to be determined. Using site-directed mutagenesis and peptide substrate structure-activity studies, we have identified specific active-site residues within rat GGTase-I involved in substrate recognition and developed novel GGTase-I variants with expanded/altered substrate selectivity. The ability to drastically alter GGTase-I selectivity mirrors similar behavior observed in FTase but employs mutation of a distinct set of structurally homologous active-site residues. Our work demonstrates that tunable selectivity may be a general phenomenon among multispecific enzymes involved in posttranslational modification and raises the possibility of variable substrate selectivity among GGTase-I orthologues from different organisms. Furthermore, the GGTase-I variants developed herein can serve as tools for studying GGTase-I substrate selectivity and the effects of prenylation pathway modifications on specific proteins.",
author = "Gangopadhyay, {Soumyashree A.} and Losito, {Erica L.} and Hougland, {James L}",
year = "2014",
month = "1",
day = "21",
doi = "10.1021/bi4011732",
language = "English (US)",
volume = "53",
pages = "434--446",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "2",

}

TY - JOUR

T1 - Targeted reengineering of protein geranylgeranyltransferase type i selectivity functionally implicates active-site residues in protein-substrate recognition

AU - Gangopadhyay, Soumyashree A.

AU - Losito, Erica L.

AU - Hougland, James L

PY - 2014/1/21

Y1 - 2014/1/21

N2 - Posttranslational modifications are vital for the function of many proteins. Prenylation is one such modification, wherein protein geranylgeranyltransferase type I (GGTase-I) or protein farnesyltransferase (FTase) modify proteins by attaching a 20- or 15-carbon isoprenoid group, respectively, to a cysteine residue near the C-terminus of a target protein. These enzymes require a C-terminal Ca1a2X sequence on their substrates, with the a1, a2, and X residues serving as substrate-recognition elements for FTase and/or GGTase-I. While crystallographic structures of rat GGTase-I show a tightly packed and hydrophobic a2 residue binding pocket, consistent with a preference for moderately sized a2 residues in GGTase-I substrates, the functional impact of enzyme-substrate contacts within this active site remains to be determined. Using site-directed mutagenesis and peptide substrate structure-activity studies, we have identified specific active-site residues within rat GGTase-I involved in substrate recognition and developed novel GGTase-I variants with expanded/altered substrate selectivity. The ability to drastically alter GGTase-I selectivity mirrors similar behavior observed in FTase but employs mutation of a distinct set of structurally homologous active-site residues. Our work demonstrates that tunable selectivity may be a general phenomenon among multispecific enzymes involved in posttranslational modification and raises the possibility of variable substrate selectivity among GGTase-I orthologues from different organisms. Furthermore, the GGTase-I variants developed herein can serve as tools for studying GGTase-I substrate selectivity and the effects of prenylation pathway modifications on specific proteins.

AB - Posttranslational modifications are vital for the function of many proteins. Prenylation is one such modification, wherein protein geranylgeranyltransferase type I (GGTase-I) or protein farnesyltransferase (FTase) modify proteins by attaching a 20- or 15-carbon isoprenoid group, respectively, to a cysteine residue near the C-terminus of a target protein. These enzymes require a C-terminal Ca1a2X sequence on their substrates, with the a1, a2, and X residues serving as substrate-recognition elements for FTase and/or GGTase-I. While crystallographic structures of rat GGTase-I show a tightly packed and hydrophobic a2 residue binding pocket, consistent with a preference for moderately sized a2 residues in GGTase-I substrates, the functional impact of enzyme-substrate contacts within this active site remains to be determined. Using site-directed mutagenesis and peptide substrate structure-activity studies, we have identified specific active-site residues within rat GGTase-I involved in substrate recognition and developed novel GGTase-I variants with expanded/altered substrate selectivity. The ability to drastically alter GGTase-I selectivity mirrors similar behavior observed in FTase but employs mutation of a distinct set of structurally homologous active-site residues. Our work demonstrates that tunable selectivity may be a general phenomenon among multispecific enzymes involved in posttranslational modification and raises the possibility of variable substrate selectivity among GGTase-I orthologues from different organisms. Furthermore, the GGTase-I variants developed herein can serve as tools for studying GGTase-I substrate selectivity and the effects of prenylation pathway modifications on specific proteins.

UR - http://www.scopus.com/inward/record.url?scp=84892708883&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84892708883&partnerID=8YFLogxK

U2 - 10.1021/bi4011732

DO - 10.1021/bi4011732

M3 - Article

C2 - 24344934

AN - SCOPUS:84892708883

VL - 53

SP - 434

EP - 446

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 2

ER -