So far, no receptor sequences for buffalo GM-CSF have been reported. Therefore, we used human GM-CSF receptors (PDB code: 3CXE; chain:A and chain:C) to build bGM-CSF and human receptor (bGMR) complex (Fig. 4). The bGMR and hGMR complexes were analysed using InterProSurf server and PIC server (Tina et al., 2007). The result is shown in Tables 1 and 2. Sixteen interface residues (VAL 16, ASN 17, ILE 19, GLN 20, GLU21, ARG23, LEU25, LEU 49, LYS72, GLY75, PRO 76, MET 79, LEU 114, LEU 115, ILE 117 and PRO 118) were identified in hGM-CSF, whereas only 10 interface residues (VAL 33, ALA 35, LEU42, SER88, GLY 91, SER92, MET 95, PHE 131, ILE 133 and PRO 134) were identified in bGM-CSF. Accessible surface analysis shows that most of the residues in contact have accessible surface area in the range of 0–20%. This indicates that the interacting residues are buried, whereas a fraction of residues are exposed. The important interactions identified as crucial for maintaining of the three-dimensional structure of a GMR complex are hydrogen bonds and hydrophobic forces. As seen in Table 1, hGMR complex has more number of hydrophobic interaction and hydrogen bonds than bGMR complex. This indicates that hGMR complex is more stable than bGMR complex.
Alpha receptor has four crucial interface residues (Leu 246, Tyr 248, Arg 302 and Ile 303) that interact with GM-CSF. Our analysis shows that both GM-CSFs establish similar interactions with those four crucial residues (Tables 1 and 2). The crucial residues in beta receptor that play role in GM-CSF interaction are Ser 102, Val 104 and Val 105. In hGMR complex, Val 104 forms hydrophobic interaction with Lue 25 (helix 1), pro 76 (helix 4) of hGM-CSF and in bGMR complex, Val 104 interacts with Leu 42 (helix 1) of bGM-CSF. It also makes hydrogen bond with Glu 38 in helix 1 of bGM-CSF. Val 105 forms hydrophobic interactions with Pro 76 (helix 1), Met 79 (helix 4) in hGM-CSF and Ala 35 (helix 1) and Met 95 (helix 4) of bGM-CSF. In addition, Val 105 form hydrogen bonds with Glu residues (helix 1) of both GM-CSFs. Lys 72 (helix 4) of hGM-CSF forms a salt bridge with Asp 107 of beta receptor. This salt bridge is absent in bGM-CSF where Lys is replaced by Ser. A salt bridge is observed between Lys 37 (helix 1) of bGM-CSF and Asp 250 of alpha receptor. This salt bridge is missing in hGM-CSF due to the absence of Lys, which is replaced by Gln.
From our analysis, it is observed that helix 4 of GM-CSF plays a role in beta receptor interaction, whereas helix 5 plays a role in alpha receptor interaction. Helix 1 of GM-CSF interacts with both alpha and beta receptors. The previous study showed that bovine GM-CSF has weak activity in both humans and mice (Charles et al., 1988). As bovine and buffalo GM-CSFs share 92% sequence identity, we can also expect a weak bGM-CSF activity in humans. It is obvious from our analysis that most of the interface residues that play a role in receptor binding are conserved in both human and buffalo GM-CSFs. Very similar interactions were observed in both hGMR and bGMR complexes. This indicates that bGM-CSF might interact with human GM-CSF receptors.