ESI-MS and FTIR studies of the interaction between the second PDZ domain of hPTP1E and target peptides.
The specificity of interaction between the second PDZ domain of human protein tyrosine phosphatase1E (PDZ2) and a C-terminal peptide, ENEQVSAV, from the guanine nucleotide exchange factor RA-GEF-2 was investigated using Fourier transform infrared (FTIR) spectroscopy and electrospray ionization mass spectrometry (ESI-MS). Specificity of the binding interaction and the importance of Ser in the -2 position of the target peptide were demonstrated using alternate peptides ENEQVCAV and KDDEVYYV. FTIR-monitored thermal denaturation in the amide I region showed a 10 degrees C increase in melting temperature (Tm) for the PDZ2-ENEQVSAV complex compared with that of free PDZ2, and the spectra revealed increased absorption in the beta-sheet region (1628 cm(-1)) of PDZ2 on peptide binding. Neither of these results were observed with peptides containing either Cys or Tyr in the -2 position. Complex formation with the Ser-containing peptide was further demonstrated by direct measurement of a 1:1 PDZ-peptide complex by ESI-MS in 100% aqueous solutions without the need for organic co-solvents. Our results demonstrate that even a single atom (O --> S) substitution from Ser to Cys in the -2 position disrupts C-terminal peptide binding to PDZ2.
Papp
Robert
R
Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West, Montreal, Quebec, Canada H3G 1M8.
Ekiel
Irena
I
English
Ann M
AM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
D020662:Guanine Nucleotide Exchange Factors; D010455:Peptides; C485746:RAPGEF6 protein, human; D011994:Recombinant Proteins; C516742:PTPN13 protein, human; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13; D017027:Protein Tyrosine Phosphatases