Capping motifs stabilize the leucine‐rich repeat protein PP32 and rigidify adjacent repeats

Abstract

Capping motifs are found to flank most b-strand-containing repeat proteins. To better understand the roles of these capping motifs in organizing structure and stability, we carried out folding and solution NMR studies on the leucine-rich repeat (LRR) domain of PP32, which is composed of five tandem LRR, capped by a-helical and b-hairpin motifs on the N-and C-termini. We were able to purify PP32 constructs lacking either cap and containing destabilizing substitutions. Removing the C-cap results in complete unfolding of PP32. Removing the N-cap has a much less severe effect, decreasing stability but retaining much of its secondary structure. In contrast, the dynamics and tertiary structure of the first two repeats are significantly perturbed, based on 1 H-15 N relaxation studies, chemical shift perturbations, and residual dipolar couplings. However, more distal repeats (3 to C-cap) retain their native tertiary structure. In this regard, the N-cap drives the folding of adjacent repeats from what appears to be a molten-globule-like state. This interpretation is supported by extensive analysis using core packing substitutions in the full-length and N-captruncated PP32. This work highlights the importance of caps to the stability and structural integrity of b-strand-containing LRR proteins, and emphasizes the different contributions of the N-and C-terminal caps.

Highly polarized C-terminal transition state of the leucine-rich repeat domain of PP32 is governed by local stability

“…This trend (at pD 6.7) is also observed for pD 6.2 and pD 7.2, indicating that exchange is limited by stability, and not by opening kinetics. Therefore, the C-terminal region appears to have much a higher local stability than the N-terminal region, consistent with our previous findings that PP32 completely unfolds upon removal of part of the C-terminal cap, but retains C-terminal structure upon removal the N-terminal α-helical cap (25). In addition, there appears to be substantial variation of protection factors within each repeat.…”

“…If PP32 folds via a polarized C-terminal pathway, removing the N-terminal region should not affect the first step of folding. To test this prediction, we measured folding kinetics of a PP32 construct that lacks the N-terminal α-helical capping motif (Δ NCap PP32) (25). The urea-dependent refolding phases of Δ NCap PP32 are similar to those of WT PP32 (Fig.…”

“…a C-terminally destabilized PP32 variant, Y131F/D146L (YD). Because the structure of YD is highly similar to that of WT PP32 (25), the destabilizing effect should be localized to the C terminus. Although YD is significantly destabilized, it retains sufficient stability for Φ-value analysis (Fig.…”

“…In contrast, HX studies observe unfolding reactions in the context of the folded state, such that structural modules (foldons) (47) can stabilize one another. Work on consensus α-helical repeat proteins (48,49) and on deletion studies of LRR proteins (25,50) have shown that the interfacial interactions between repeats are strongly stabilizing. Thus, we expect the highly stable C terminus of PP32 to protect neighboring repeats from exchange, broadening the observed stability map toward the N terminus.…”

“…The N and C termini of PP32 are capped by a helix-loop-helix motif and a β-hairpin, respectively. Deletion and mutational analysis showed that whereas the N-terminal cap contributes stability, the C-terminal cap is critical for stability: without the C-terminal cap, PP32 is entirely unfolded (25). Here we investigate the effects of destabilizing substitutions on folding kinetics and find that PP32 folds via an on-pathway intermediate with a highly polarized C-terminal pathway.…”