Wp. Bocchinfuso et Gl. Hammond, STEROID-BINDING AND DIMERIZATION DOMAINS OF HUMAN SEX HORMONE-BINDINGGLOBULIN PARTIALLY OVERLAP - STEROIDS AND CA2+ STABILIZE DIMER FORMATION, Biochemistry, 33(35), 1994, pp. 10622-10629
Human sex hormone-binding globulin (SHBG) is a homodimeric plasma glyc
oprotein with a single steroid-binding site for biologically active se
x steroids, and a methionine at position 139 (M139) interacts with the
photoaffinity ligand, Delta(6)-testosterone. We have introduced amino
acid substitutions into this and other locations in the SHBG molecule
and have examined their impact on steroid binding and dimerization. A
s a result, substitutions at residues 134-139 generate alterations in
steroid-binding specificity. In particular, substitutions at residues
134-138 were characterized by altered binding affinities for estradiol
relative to 5 alpha-dihydrotestosterone (DHT), and one of them (R135L
) also showed a 2-fold increase in affinity for C19 steroids with a 3
beta-hydroxy group. Unlike all of the other mutants we have examined,
the M139W mutant has a 5-fold lower affinity for DHT, and its affiniti
es for testosterone, 5 alpha-androstane-3 beta,17 beta-diol, and estra
diol also appear to be reduced to a similar extent. By contrast, M139W
appears to bind androst-5-ene-3 beta,17 beta-diol with only 2-fold le
ss affinity than wild-type SHBG, while its affinity for 19-nortestoste
rone remains unaffected. Substitutions at other positions, including t
hose immediately C-terminal to M139, had no effect on steroid-binding
affinity and/or specificity. These data provide evidence that residues
134-139 influence the recognition of specific A/B ring conformations
of steroid ligands and may constitute part of the steroid-binding doma
in. We have also found that substitutions at residues 138-148 impair d
imerization and that this defect may be abrogated by occupancy of the
steroid-binding site. The removal of divalent cations also destabilize
s dimer formation of mutants with substitutions at residues 140-148, a
nd Ca2+ or Zn2+, but not Mg2+, can restore their ability to form dimer
s. Steroid ligands and divalent cations also appear to act independent
ly to stabilize dimer formation. These data lead us to conclude that t
he steroid-binding and dimerization domains of human SHBG partially ov
erlap and that steroid ligands and divalent cations influence the dime
rization interface to enhance subunit association.