INTRODUCTION
1 Organisation of human NOS domains. The annotation is based on polypeptide sequence of human nNOS, eNOS and iNOS. Human iNOS and human eNOS have very similar domain organisations from the N terminal to the C terminal. Human nNOS has an additional extracellular N terminal PDZ domain (shown in green) and a transmembrane helix compared to eNOS and iNOS. Zinc binding sites, oxygenase domain with HEM, substrate L-Arginine and H4B binding sites, reductase region with FMN, FAD and NADPH binding sites of these three human NOS isoforms are presented. Lipid modification of human eNOS can occur (palmitoylation at Cys15 and Cys26, and Myristoylation at Gly2) that facilitates its anchor onto the membrane. Both Human nNOS and eNOS have auto-inhibitory elements which human iNOS lacks. Regulatory Calmodulin is shown by “CaM” near the CaM binding sites |
RESULTS
Characteristics of bound compounds
2 Scatter plot showing molecular weights, binding interface areas, and theoretical standard binding free energies (ΔG) of inhibitory compounds bound to human eNOS and nNOS. A Human eNOS. The binding interface area is positively correlated with molecular weight, while negative correlation dominates the relationship between theoretical binding energy value and binding interface area. The binding energy of HEC (an heme analogue, 3EAH.pdb) is omitted as it’s beyond the current scale. For 8EY (5UOA.pdb), M4R (5UOB.pdb), KMM (6NH7.pdb), KL4 (6NH3.pdb) and M16 (6POU.pdb), only one copy of the compound molecule which is in the active site is included. B Human nNOS. Neither binding interface area nor molecular weight is correlated to the value of theoretical binding energy in a consistent fashion. C Binding interface area is not consistently positively correlated to the absolute value of theoretical binding energy for human nNOS |
3 Plot of ClogPs (molar refractivity, partition coefficient) and theoretical binding energies of human eNOS- and nNOS-bound compounds. A For eNOS, the theoretical binding energy value is negatively correlated with hydrophobicity or lipophilicity (represented by theoretical CLogP values) of the compound, which means lipophilicity constructively contributes to the energetically favored heat-releasing binding to eNOS. B No such pattern is observed for human nNOS |
Attributes of the active sites of human eNOS, nNOS and iNOS
4 Statistical summary of the interaction frequency of human NOS residues with inhibitors or H4B. X-axis is amino acid residues of human NOS, and Y-axis is the involvement frequency of the residue in compound binding. A Human eNOS. Although there are residues which interact with both inhibitors and H4B, like Val104 and Arg365, distinct binding regions of inhibitors and H4B can be observed. This is consistent with Fig. 5. The binding frequencies of residues to H4B are normalized to 28 with a threshold of 15. B Human nNOS |
5 Binding profiles of inhibitors, H4B, HEM and substrate arginine to human eNOS, human nNOS and human iNOS. A Stereo view of the superposed active site of eNOS structures with inhibitors shown in thin lines, and H4B and HEM shown in sticks. B Surface capping views of the human eNOS active site. HEM is shown in thin lines; H4B and substrate arginine are shown in sticks. The slight hydrophobicity of both the arginine binding site and the H4B binding site can be observed, which is consistent with Fig. 3A and Fig. 6. The inhibitor binds to the substrate arginine-binding pocket, and this is far away from the H4B binding site. C Stereo view of the superposed active site of nNOS structures with inhibitors shown in thin lines, and H4B and HEM shown in sticks. D Surface capping views of the human nNOS active site. HEM, H4B and substrate arginine are shown in thin lines. The charge distribution and hydrophobicity (or hydrophilicity) properties of both the arginine-binding site and the H4B-binding site are similar to that of human eNOS. E Stereo view of the superposed active site of iNOS structures with inhibitors shown in thin lines, and H4B and HEM shown in sticks. F Surface capping views of the human iNOS active site (1NSI.pdb) |
6 Sequence alignment of human eNOS and human nNOS based on structure superimposition. eNOS monomer from 4d1o.pdb and nNOS monomer from 6av3.pdb are superposed and aligned by using UCSF Chimera (Pettersen et al. 2004) and ESPript (Robert and Gouet 2014). Human eNOS residues Val104, Phe105, Asn366, Pro370, His371, which corresponds to human nNOS Met341, His342, Asp602, Asn606, Ser607, are highlighted by green triangles. Among these residues, eNOS-Asn366 and nNOS-Asp602 are within the substrate arginine binding pocket, and Val104, Phe105, Pro370, His371, Met341, His342, Asn606, Ser607 are located at the opening of the cavity. RMSD between human eNOS (4d1o.pdb) and human nNOS (6av3.pdb) monomers is ~0.61 Å |
Mechanism of action
1 Interface areas between H4B and human NOS monomer, between H4B and HEM, and between two polypeptide chains from NOS dimers |
Human NOS | Interface area between H4B and one monomer (Å2) | Interface area between H4B and the other monomer (Å2) | Interface area between H4B and HEM from one monomer (Å2) | Interface area between polypeptide chain from monomer A and monomer B (Å2) |
eNOS | 212 | 119 | 45 | 2661 |
nNOS | 192 | 116 | 40 | 2683 |
iNOS | 209 | 118 | 44 | 2690 |
DISCUSSION
9 Sequence alignment of human iNOS and human nNOS based on structure superimposition (between iNOS monomer from 3e7g.pdb and nNOS monomer from 6av3.pdb). Human iNOS residues Thr121, Ala262, Asn370, Val386, Gln387, which correspond to human nNOS His342, Ser482, Ser590, Asn606 and Ser607, are highlighted by green triangles. These residues are located at the entrance of the substrate (or inhibitor) binding pocket. RMSD between human iNOS (3e7g.pdb) and human nNOS (6av3.pdb) monomers is ~0.63 Å |