Synthetic peptides as structural maquettes of Angiotensin-I converting enzyme catalytic sites

Abstract

The rational design of synthetic peptides is proposed as an efficient strategy for the structural investigation of crucial protein domains difficult to be produced. Only after half a century since the function of ACE was first reported, was its crystal structure solved. The main obstacle to be overcome for the determination of the high resolution structure was the crystallization of the highly hydrophobic transmembrane domain. Following our previous work, synthetic peptides and Zinc(II) metal ions are used to build structural maquettes of the two Zn-catalytic active sites of the ACE somatic isoform. Structural investigations of the synthetic peptides, representing the two different somatic isoform active sites, through circular dichroism and NMR experiments are reported.

Figure 1

Synthetic peptide maquettes of the N- and C- active site domains of human somatic ACE (sACE). Sequence numbering of peptides, sACE, testis isoform (tACE) and crystal structures domains. The different residues among the two sequences are highlighted.

Figure 2

(a) Short-, medium-, and long-range connectivities. (b) Number of NOE constraints per residue (white, gray, dark gray, and black vertical bars represent, resp., intraresidue, sequential, medium-range and long-range connectivities). (c) Schematic representation of the sequential and medium range NOEs involving HN, Hα, and H_β protons for Zn²⁺-ACE_N(37) (corresponds to His³⁶⁰-Ala³⁹⁶ of the human somatic form).

Figure 3

(a) Short-, medium-, and long-range connectivities. (b) Number of NOE constraints per residue (white, gray, dark gray, and black vertical bars represent, resp., intraresidue, sequential, medium-range, and long-range connectivities). (c) Schematic representation of the sequential and medium range NOEs involving HN, Hα, and H_β protons for Zn²⁺-ACE_C(37) (corresponds to His⁹⁵⁸-Ala⁹⁹⁴ of the human somatic form).

Figure 4

Circular dichroism spectra (left) and corresponding diagrams (right) of helical content through data analysis by CDNN software of (a) 2,2,2-trifluoroethanol (TFE) range from 0% to 100% of Zn²⁺-ACE_N(37) samples, at pH = 5.0, T = 25°C, 50 mM Tris-HCl, and 200 mM NaCl and (b) of pH range from 2.6 to 7 of Zn²⁺-ACE_N(37) samples, at 65% TFE, T = 25°C, 50 mM Tris-HCl, and 200 mM NaCl.

Figure 5

Fingerprint regions of 600 MHz TOCSY ((a) ACE_C(37) and (b) ACE_N(37)) and NOESY ((c) ACE_C(37) and (d) ACE_N(37)) spectra recorded at T = 298 K. The sequential connectivity pattern shown indicates the peptide sequence-specific resonance assignment.

Figure 6

(a) Ensemble of DYANA 30 best models of the Zn²⁺-ACE_N(37) (corresponds to His³⁶⁰-Ala³⁹⁶ of the human somatic form) calculated with NMR data. (b) Ribbon diagram of Zn²⁺-ACE_N(37) peptide.

Figure 7

(a) Ensemble of DYANA 30 best models of the Zn²⁺-ACE_C(37) (corresponds to His⁹⁵⁸- Ala⁹⁹⁴of the human somatic form), calculated with NMR data. (b) Ribbon diagram of Zn²⁺-ACE_C(37) peptide.

Figure 8

Backbone and ribbon representation of the solution structures of both Zn²⁺-ACE_N(37) (a) (corresponds to His³⁶⁰-Ala³⁹⁶ of the human somatic form) and Zn²⁺-ACE_C(37) (b) (corresponds to His⁹⁵⁸- Ala⁹⁹⁴of the human somatic form).

Figure 9

Superimposition of the crystal structure (in red) and the solution structure derived from NMR data (in cyan) of Zn²⁺-ACE_N(37) peptide (a) and Zn²⁺-ACE_C(37) peptide (b).