Figure 5. Characterization of the BAFF and TACI interaction.
a, Binding of TACI CRD1 (residues 14 -69) and CRD2 (residues 67
-111) to BAFF. The conserved Asp and Leu residues of the DxL
motifs are mutated to alanines in the mutants (M). The TACI FL
is the extracellular domain (residues 1 -156) of the full-length
TACI. After the binding assay, the resulting duplicate SDS-PAGE
gels were visualized either by Coomassie staining or by
immunoblot analysis using a polyclonal antibody specific to BAFF
(Upstate Biotech) (see Methods). b, Interaction of the TACI CRDs
with BAFF. C1M contains mutations of Asp41 and Leu43 of the
first DxL motif to alanines. C2M contains mutations of Asp80 and
Leu82 of the second DxL motif to alanines. C1,2M contains
simultaneous double alanine mutations of both DxL motifs of TACI
FL. The resulting duplicate SDS-PAGE gels were visualized as in
a. c, CRD1 and CRD2 share the same binding site as BAFF. BAFF
has a deletion of the DE loop. The R265A BAFF DE
mutant has a mutation of Arg265 to alanine and a deletion of the
DE loop. C1M, C2M and C1,2M are TACI mutants, as described in b.
The resulting duplicate SDS-PAGE gels were visualized as in a.
The flexible His tag of the BAFF DE
mutant was partially digested during purification and runs as a
diffuse band in the SDS-PAGE gel. d, Model of the TACI
extracellular domain bound to BAFF. The TACI extracellular
domain (residues 32 -104) is shown in a broken black line.
Negatively and positively charged surfaces of BAFF are colored
red and blue, respectively. The N and C termini of the TACI
extracellular domain are indicated. Viewing orientation is
similar to that of Fig. 3b. A1 and C2 modules of CRDs are
indicated. The structures of the A1 modules were built with that
of BAFF-R CRD as a template. The structures of the C2 modules
and the angles between the A1 and C2 modules were adopted from
the structure of TNFR1 CRD4 for model building. The molecular
modeling was carried out using WHATIF^43.
The above figure is reprinted
by permission from Macmillan Publishers Ltd:
Nat Struct Biol