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

Abstract

The leucine-rich repeat domain of PP32 is composed of five β-strand-containing repeats anchored by terminal caps. These repeats differ in sequence but are similar in structure, providing a means to connect topology, sequence, and folding pathway selection. Through kinetic studies of PP32, we find folding to be ratelimited by the formation of an on-pathway intermediate. Destabilizing core substitutions reveal a transition state ensemble that is highly polarized toward the C-terminal repeat and cap. To determine if this nucleus for folding corresponds to the most stable region of PP32, we monitored amide hydrogen exchange by NMR spectroscopy. Indeed, we find the highest protection to be biased toward the C terminus. Sequence manipulations that destabilize the C terminus spread out the transition state toward the middle of the protein. Consistent with results for helical ankyrin repeat proteins, these results suggest that local stabilities determine folding pathways.A lmost 50 y ago, Cyrus Levinthal suggested that protein folding is a guided process, rather than a random search (1). Since then, it has been proposed that rapid folding can be accomplished via a single preferred pathway (2-4). However, contrasting models of funneled, nonspecific folding capture key aspects of the folding process, including specific intermediates and transient stable structures (5, 6). To what extent does protein folding follow a single pathway, and to the extent that a single pathway dominates, what determines that pathway?For some globular proteins, specific kinetic intermediates have been identified with features of thermodynamically stable substructures of the native state. This observation suggests specific pathways that correspond to low energy folding routes (7-12). However, directly testing this thermodynamic control of folding pathway selections requires mapping of local stability. Hydrogen exchange methods have provided the most detailed energy maps of native proteins (13), but sequence-distant contacts and irregular tertiary structures of globular proteins tend to blur the boundaries of local stability.In contrast to globular proteins, elongated repeat proteins comprise tandem repeated secondary structural units, giving rise to regular topology lacking sequence-distant contacts. As a result, the contributions made by different regions of a repeat protein to its folding thermodynamics and kinetics can be easily dissected and compared. If local stability dictates folding, a preferred pathway should be observed, because repeats differ in sequence. If topology drives folding, multiple parallel pathways would be observed, because the repeats are similar in structure. For designed consensus α-helical ankyrin repeat constructs, with repeats of nearly identical sequence, folding proceeds via parallel pathways (14). In contrast, naturally occurring ankyrin repeat proteins with repeats of high sequence variation fold through preferred pathways (15-18). Regions that initiate folding correspond to low-energy structures, based on energy ...

The consequences of cavity creation on the folding landscape of a repeat protein depend upon context

“…S2 E-J, S3 E-J, and S4 E-J and Table S1), indicating that all residues reflect the complete transition from the folded to the unfolded state. The average ΔG f values are in good agreement with the values obtained for these variants previously from CD-detected urea unfolding (Table 1) (21). The volume changes for these variants are much larger (by 59-72 mL/mol) than that obtained previously for WT pp32 (Table 1 and SI Appendix, Fig.…”

“…1). The global stability and folding mechanism of WT pp32 have been well characterized (21,22). The ϕ-value analysis revealed an early transition state in which the C-terminal capping motif and the fifth repeat are ordered at the folding barrier.…”

“…We investigate here the effects of cavity creation on the folding landscape of pp32 using five of the cavity-containing variants engineered previously for ϕ-value analysis (21). These variants harbor cavities in the N-terminal capping motif (I7A), and the second (L60A), third (L83A), fourth (L109A), and fifth (L139A) repeats ( Fig.…”

The case for defined protein folding pathways

“…The repeat units are individually cooperative, have low stability, and are further stabilized by their interfacial interactions. Repeat proteins have been found to fold in repeatsized steps, one or several units at a time, in a distinct pathway order (66,67)…”

Identification of Novel Mutations in the LRR-Cap Domain ofC21orf2in Japanese Patients With Retinitis Pigmentosa and Cone–Rod Dystrophy

“…It was of great interest to us that a kinetic study 27 has shown that LRRCT is an essential core domain required for proper folding of the LRR protein PP32. Since the replacement of the first tyrosine or the fifth valine of the LRRCT consensus sequence affects the folding of LRR protein, 10,27 missense mutations in these amino acids were expected to decrease the levels of functional protein. We preferentially studied the compound heterozygous mutations to confirm the mutation impacts on both Y107 and V111.…”