Correlating structure and affinity for PEX5:PTS1 complexes.
Many proteins that are destined to reside within the lumen of the peroxisome contain the peroxisomal targeting signal-1 (PTS1), a C-terminal tripeptide approximating the consensus sequence -Ser-Lys-Leu-COO(-). The PTS1 is recognized by the tetratricopeptide repeat (TPR) domains of PEX5, a cytosolic receptor that cycles between the cytoplasm and the peroxisome. To gain insight into the energetics of PTS1 binding specificity and to correlate these with features from the recently determined structure of a PEX5:PTS1 complex, we used a fluorescence-based binding assay that enables the quantitation of the dissociation constants for PTS1-containing peptide complexes with the TPR region of human PEX5. Through application of this assay to a collection of pentapeptides containing different C-terminal tripeptide sequences, including both natural and unnatural amino acids, the thermodynamic effects of sequence variation were examined. PTS1 variants that correspond to known functional targeting signals bind to the PEX5 fragment with a change in the standard binding free energy within 1.8 kcal mol(-1) of that corresponding to the peptide ending with -Ser-Lys-Leu-COO(-). The results suggest that a binding energy threshold may determine the functionality of PTS1 sequences.
Gatto
Gregory J
GJ
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Maynard
Ernest L
EL
Guerrerio
Anthony L
AL
Geisbrecht
Brian V
BV
Gould
Stephen J
SJ
Berg
Jeremy M
JM
D019943:Amino Acid Substitution / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D005454:Fluorescence Polarization / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D005456:Fluorescent Dyes; D046911:Macromolecular Substances; C000614264:PEX5 protein, human; D010446:Peptide Fragments; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D012694:Serine