Biology Reference
In-Depth Information
domain D2, Phe208 also makes contacts with the hydrophobic pocket. Two
electrostatic interactions occur between the
-helix and other regions of
EPOR. Several salt bridges function to link the
α
-helix with D1 and D2,
including the electrostatic interactions of Lys14 with Glu31 (on domain D1)
and Asp122 (on domain D2).
Alteration of the WSXWS sequence disrupts ligand binding and receptor
signaling. The WSXWS motif has been shown to be critical for the folding and
transport of the receptor to the cell surface, and an A211E mutation further
improved the efficiency of the processes. In the structure of an rHuEPO-EPOR
complex, Glu211 occupies the X position. The side-chain of Glu211 in the
WSXWS motif is closest (<4.5 Å) to Leu17 of the
N
-terminal receptor helix.
N
Additionally, the side chains of the tryptophans in the WSXWS motif, residues
Trp209 and Trp212, sandwich the hydrophobic side chain of Arg197 in the
receptor fold,while Ser210 and Ser213 are within hydrogen-bonding distance
of Ala198 and Val196, respectively. The observation that the WSXWS motif
interacts with with the
N
-terminal helix and the
N
α
-sheet residues (Val196,
Arg197, and Ala198) in the rHuEPO-EPOR complex, suggests that the
N
-ter-
N
minal helix may be important in stabilizing the folded EPOR through an inter-
action with WSXWS. The relationship of WSXWS, the
β
-helix, therefore,
may be essential in holding D1 and D2 in the precise geometry allowing
dimerization and signal transduction of the ligand.
The rHuEPO-EPOR complex is a 2:1 stoichiometry, and the receptor mol-
ecules are held together through two distinct surface areas on rHuEPO that are
located on opposing faces of the molecule. These rHuEPO/EPOR interfaces
have been identified as high-affinity (Kd approximately 1 nM) and low-affin-
ity (Kd approximately 1 µM) binding sites and are referred to as site 1 and site
2, respectively. Site 1 comprises rHuEPO residues from helices A, B', D, and
part of the AB loop; site 2 comprises the A and C helices, exclusively.
Presumably site 1 binds more tightly than site 2 because of the larger number
of residues participating in the interaction of the first site compared with the
second site. Site 1 is characterized by a hydrophobic-binding region in the
middle of the binding site with flanking amino acids that allow electrostatic
interactions with EPOR. EPOR loops L1, L5, and L6 interact with the
hydrophobic rHuEPO residues with Phe93 of EPOR as the dominant
hydrophobic contact. Phe93 is a pivotal residue firmly held in place by hydro-
gen bonding to rHuEPO residues Thr44 and Asn147. Mutagenesis has shown
that Phe93 on the receptor is a critical rHuEPO-binding determinant; and
mutation of this residue eliminates any detectable binding [18]. The site-1
binding site of rHuEPO also has contributions from the A-B linker polypeptide
residues (Thr44-Phe48) of rHuEPO. The hydrophobic binding surface of site
1 itself is surrounded by hydrophilic interactions with Asn147 of rHuEPO
functioning as a major contributor to the electrostatic interactions. Asn147
binds to EPOR using three hydrogen bonds, one of which is between its N
α
δ
atom and the carbonyl oxygen of Phe93. On either side of the Asn147 in EPO
