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.