In an effort to gain greater insight into the evolution of the redox a
ctive,
catalyti
c antibody28B4, the germline genes used by the mouse to generate this antibody were
cloned and expressed, andthe X-ray
crystal stru
ctures of the unliganded and hapten-bound germline Fab of antibody 28B4 weredetermined. Comparison with the previously determined stru
ctures of the unliganded and hapten-boundaffinity-matured Fab [Hsieh-Wilson, L. C., S
chultz, P. G., and Stevens, R. C. (1996)
Proc. Natl. Acad.Sci. U.S.A. 93, 5363] shows that the germline antibody binds the
p-nitrophenyl ring of hapten
3 in anorientation signifi
cantly different from that seen in the affinity-matured antibody, whereas the phosphonatemoiety is bound in a similar mode by both antibodies. The affinity-matured antibody 28B4 has moreele
ctrostati
c and hydrophobi
c intera
ctions with hapten
3 than the germline antibody and binds the haptenin a lo
ck-and-key fashion. In
contrast, signifi
cant
conformational
changes o
ccur in the loops of CDR H3and CDR L1 upon hapten binding to the germline antibody,
consistent with the notion of stru
ctural plasti
cityin the germline antibody-
combining site [Wedemayer, G. J., Patten, P. A., Wang, L. H., S
chultz, P. G.,and Stevens, R. C. (1997)
Science 276, 1665]. The stru
ctural differen
ces are refle
cted in the differentialbinding affinities of the germline Fab (
Kd = 25
M) and 28B4 Fab (
Kd = 37 nM) to hapten
3. Ninerepla
cement mutations were found to a
ccumulate in the affinity-matured antibody 28B4
compared to itsgermline pre
cursor. The effe
cts of ea
ch mutation on the binding affinity of the antibody to hapten
3 were
chara
cterized in detail in the
contexts of both the germline and the affinity-matured antibodies. One ofthe mutations, Asp95
HTrp, leads to a
change in the orientation of the bound hapten, and its presen
ce isa prerequisite for other somati
c mutations to enhan
ce the binding affinity of the germline antibody forhapten
3. Thus, the germline antibody of 28B4 a
cquired fun
ctionally important mutations in a stepwisemanner, whi
ch fits into a multi
cy
cle mutation, affinity sele
ction, and
clonal expansion model for germlineantibody evolution. Two other antibodies, 20-1 and NZA6, with very different antigen spe
cifi
cities werefound to be highly homologous to the germline antibody of 28B4,
consistent with the notion that
certaingermline variable-region gene
combinations
can give rise to polyspe
cifi
c hapten binding sites [Romesberg,F. E., Spiller, B., S
chultz, P. G., and Stevens, R. C. (1998)
Science 279, 1929]. The ultimate spe
cifi
cityof the polyspe
cifi
c germline antibody appears to be defined by CDR H3 variability and subsequent somati
cmutation. Insights into the evolution of antibody-
combining sites provided by this and other stru
cturalstudies are dis
cussed.