Biophysical studies of membrane proteins/peptides. Interaction with ...

BINDING OF INHIBITORS TO A PUTATIVE BINDING
DOMAIN OF V-ATPase
III
BINDING OF INHIBITORS TO A
PUTATIVE BINDING DOMAIN
OF V-ATPase
1. Introduction
Osteoporosis, a disease endemic in Western society, is characterized by a net loss of
bone mass which results from an imbalance of the bone-remodelling process, with bone
resorption exceeding bone formation. Bone demineralization involves acidification of
the isolated extracellular microenvironment. The lowering of pH is necessary both to
dissolve the bone material and to provide the acidic environment required by the
collagenases to degrade the bone matrix (Teitelbaum, 2000). The acidification is due to
proton translocation by the vacuolar-type H + -ATPases (V-ATPases) that are present in
large numbers on the ruffled border of the osteoclast while the counterions diffuse
passively through a chloride channel (Fig. III-1). Although there are a number of drugs
directed to osteoporosis treatment through indirect inhibition of osteoclasts (Lark and
James, 2002; Rodan and Martin, 2000), they allow only for a limited therapy as bone
loss is only temporarily reduced due to weak inhibition of these cells. The osteoclast V-
ATPase is, therefore, an attractive alternative target for the development of better
therapeutic agents that effectively and safely reduce osteoclast activity.
V-ATPases are large, multisubunit complexes organized in two domains (Fig. III.2)
(Nishi and Forgac, 2002; Kawasaki-Nishi et al., 2003). The V 1 domain, which is
responsible for ATP hydrolysis, comprises eight different subunits (A-H) that can be
removed from the membrane in soluble form. The V o domain, which is tightly
associated with the membrane and composed of five different subunits (a, c, c’, c’’, and
d), is responsible for proton translocation. The enzyme functions as a rotary motor
(Yokoyama et al., 2003; Harrison et al., 1997). By analogy with the mechanism
proposed for the F-ATPase, ATP hydrolysis at the catalytic sites drives rotation of the
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