title: Capping motifs stabilize the leucine-rich repeat protein PP32 and rigidify adjacent repeats.
abstract: Capping motifs are found to flank most β-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 α-helical and β-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-cap-truncated PP32. This work highlights the importance of caps to the stability and structural integrity of β-strand-containing LRR proteins, and emphasizes the different contributions of the N- and C-terminal caps.
authors.affiliation: T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland, 21218.
authors.forename: Thuy P
authors.initials: TP
authors.lastname: Dao
authors.forename: Ananya
authors.initials: A
authors.lastname: Majumdar
authors.forename: Doug
authors.initials: D
authors.lastname: Barrick
mesh_terms: D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism; D000090263:Leucine-Rich Repeat Proteins; D009687:Nuclear Proteins; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins
chemical_list: C087425:ANP32A protein, human; D047908:Intracellular Signaling Peptides and Proteins; D000090263:Leucine-Rich Repeat Proteins; D009687:Nuclear Proteins; D011506:Proteins; D016601:RNA-Binding Proteins
keywords: capping motifs; folding; leucine-rich repeat; packing; stability; β-strand-containing repeat proteins