Trp37 plays a key role in the interactions between the
N-terminal region and the b5 domain.
We generated two truncation mutants of N/b5 (Figure 1 ) and
examined the effects of the truncations on CD spectra. The first mutant
involved the removal of residues 1-21, the portion carrying the lowest
conservation among animals, to yield N/b5-Δ21. Residues 1-34 were also
removed to produce a domain fragment (N/b5-Δ34) analogous to the
truncated Ncb5or that was initially
cloned.6
Deleting the first 21 residues of N/b5 had no significant effect on the
near-UV CD spectrum, or on the intensity of the positive far-UV CD band
near 230 nm (Figures 2E-F, Table 1 ). The most notable effect on
CD spectra caused by removing residues 1-21 was a decrease in negative
ellipticity of the lowest wavelength far-UV band, along with a slight
red shift of that band. These observations suggest that residues 1-21
are almost exclusively disordered and show that they play no role in the
secondary and tertiary structure formation resulting from the
interactions between the N-terminal region and the b5 core. They also
support our conclusion that the near-UV CD signals and the positive
far-UV feature near 230 nm arise from Trp37, with no contribution from
Phe9.
Comparison of the far-UV CD difference spectra for N/b5-Δ21 and N/b5-Δ34
(Figure 2E, Table 1 ) shows that deleting the additional
residues 22-34 decreased, but did not abolish, secondary structure
content. In addition, Figure 2F (also Table 1 ) reveals
diminished intensity for the near-UV CD signals attributable to Trp37 in
N/b5-Δ34, but the signals in this region are much more like those in the
spectrum of N/b5 than of b5. This suggests that Trp37 is involved in
tertiary structure in both truncation mutants, but that removal of
residues 22-34 causes subtle disruption of that tertiary structure.
The evidence obtained with the truncation mutants strongly indicates a
key role for Trp37 in interactions between the N-terminal region and the
b5 domain of Ncb5or. We therefore examined the effect of mutating Trp37
to Ala on CD spectra of N/b5. Subtracting the far-UV CD spectrum of b5
from that of N/b5-W37A yielded a difference spectrum similar to the
spectrum of N-term (Figure 2G ). Moreover, the W37A mutation in
N/b5 resulted in loss of the near-UV CD features present in the N/b5
spectrum (Figure 2H ). It can therefore be concluded that the
W37A mutation in N/b5 abolishes both secondary and tertiary structure.
In comparison, the difference spectrum obtained by subtracting the
far-UV CD spectrum of N/b5-LMAA from that of N/b5 exhibits modestly
increased intensity of the negative band near 220 nm (Figure 2G,
Table 1 ) and of the positive band near 190, suggesting a small increase
in helix content. This may be attributable to the fact that Ala has a
much higher helix propensity than do Leu and
Met.41 More
notably, the LM→AA mutation does not significantly alter the shape or
intensity of the near-UV and far-UV CD features attributable to Trp37
(Figure 2G-H ). However, the CD Soret band signal for the LMAA
mutant is virtually identical to that of Ncb5or-b5 (Figure 2H ).
These observations suggest that the LM→AA mutation does not alter the
ability of N-term to dock with the b5 core, with associated adoption of
tertiary structure involving Trp37, but that it alters (or perhaps
weakens) interactions between the N-terminal region and the b5 core.
The results described in this section indicate that residues spanning
Met35 through Leu50 are sufficient to induce secondary structure (likely
helical) and accompanying tertiary structure in the N-terminal region
when it docks with the b5 core. The results also show that Trp37 plays
an essential role in inducing this structural transition, suggesting a
specific recognition site on the b5 domain surface. These results
further indicate that the induced secondary structure is propagated
toward the N-terminus but does not extend beyond Gly22. This additional
secondary structure appears to stabilize the tertiary structure.