Biology Reference
In-Depth Information
are arginine residues, Arg143 and Arg150, which form salt bridges with acids
Glu60 and Glu117 of rHuEPO, respectively.
rHuEPO site-2 interactions with EPOR are less extensive than site 1. Most
of the site-2 interactions are between residues of the C helix of rHuEPO and
the L3 loop of EPOR. The hydrophobic surface is created on EPOR by
residues Phe93, Phe205, and Met150, although because of the positioning of
the D1 domain relative to the site-2 interactions with rHuEPO, a relatively flat
EPOR surface exists that allows interaction with rHuEPO residues. The clos-
est non-polar contact in site 2 includes rHuEPO Leu108 and rHuEPOR Phe93
C
(3.9 Å). The rest of the C helix residues are positioned at a greater distance
from the EPOR hydrophobic surface than at the site-1 interface. Phe93 is again
the major contributor to the central hydrophobic-binding pocket (Leu5, Val11,
Tyr15, Ser104, Thr107, and Leu108 of EPO are within 4.5 Å of EPOR Phe93).
Met150 of EPOR, however, is buried more deeply here than in site 1, making
van der Waals contacts with Arg
10
, Va l
11
, and Arg
14
of rHuEPO.
In general, the side-chain interactions at both interfaces are predominantly
between positively charged lysines and arginines of rHuEPO and negatively
charged aspartate and glutamate side-chains of EPOR. Site 1 contains almost
γ
twice as many side-chain - side-chain interactions as site 2. The number of
hydrophilic contacts involving main-chain atoms from either rHuEPO or
EPOR, however, is equal for both sites. In addition, site 1 contains two hydro-
gen bonds that involve main-chain atoms from both rHuEPO and EPOR.
Hydrophilic interactions from three EPOR residues are common to both inter-
faces, two of which involve hydrogen bonding from a main-chain atom.
Although most residues that interact across both interfaces are polar, the
expected contribution to binding affinity is greatest from the hydrophobic con-
tacts.
Structure of a rHuEPO peptide mimetic bound to EPOR
In an elegant series of experiments, Livnah and co-workers resolved the struc-
ture of a soluble EPOR complexed with an agonistic peptide, which had no
sequence identity to the naturally occurring ligand (Fig. 4). Included in this
body of work was the bacterial expression, purification, and refolding of a sol-
uble form of EPOR (EBP) [19] and the isolation of the self-dimerizing 20
amino acid peptide EMP1 (GGTYSCHFGPLTWVCKPQGG) [9]. The peptide
has
-sheet structure and a disulfide bond constraining the peptide. The stoi-
chiometry of the EBP/EMP1 complex is one peptide homodimer bound to two
EPOR monomers. EMP1 was originally identified using peptide phage display
technology. The 20-amino acid peptide mimicked the biologic activity of
rHuEPO and functioned as an agonist. The affinity of the peptide, compared
with rHuEPO, binds approximately 1,000-fold less at 200 nM, compared with
200 pM.
β
