Incorporating β-Turns and a Turn Mimetic out of Context in Loop 1 of the WW Domain Affords Cooperatively Folded β-Sheets

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100

␤-Turns are common conformations that enable proteins to adopt globular structures, and their formation is often rate limiting for folding. ␤-Turn mimics, molecules that replace the i ؉ 1 and i ؉ 2 amino acid residues of a ␤-turn, are envisioned to act as folding nucleators by preorganizing the pendant polypeptide chains, thereby lowering the activation barrier for ␤-sheet formation. However, the crucial kinetic experiments to demonstrate that ␤-turn mimics can act as strong nucleators in the context of a cooperatively folding protein have not been reported. We have incorporated 6 ␤-turn mimics simulating varied ␤-turn types in place of 2 residues in an engineered ␤-turn 1 or ␤-bulge turn 1 of the Pin 1 WW domain, a three-stranded ␤-sheet protein. We present 2 lines of kinetic evidence that the inclusion of ␤-turn mimics alters ␤-sheet folding rates, enabling us to classify ␤-turn mimics into 3 categories: those that are weak nucleators but permit Pin WW folding, native-like nucleators, and strong nucleators. Strong nucleators accelerate folding relative to WW domains incorporating all ␣-amino acid sequences. A solution NMR structure reveals that the native Pin WW ␤-sheet structure is retained upon incorporating a strong E-olefin nucleator. These ␤-turn mimics can now be used to interrogate protein folding transition state structures and the 2 kinetic analyses presented can be used to assess the nucleation capacity of other ␤-turn mimics.beta-sheet nucleator ͉ kinetic assessment of turn mimics ͉ Pin WW domain ͉ protein folding

Section: Resultsmentioning

confidence: 99%

“…Pin WW is a three-stranded ␤-sheet protein whose spontaneous two-state folding is reversible (4,8,10,(27)(28)(29)(30)(31)(32)(33)(34)(35). Its ␤-sheet exhibits a slight right-handed twist, as revealed by crystal and solution structures (36,37).…”

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confidence: 99%

Evaluating β-turn mimics as β-sheet folding nucleators

Fuller

Liu

et al. 2009

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100

“…Pin WW variants were prepared by solid-state peptide synthesis (Ͼ 95% purity) as described elsewhere (41). FBP WW variants were expressed recombinantly and purified as described in detail elsewhere (2, 3).…”

Section: Methodsmentioning

confidence: 99%

Structure–function–folding relationship in a WW domain

Jäger

Zhang

Bieschke

et al. 2006

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211

308

Protein folding barriers result from a combination of factors including unavoidable energetic frustration from nonnative interactions, natural variation and selection of the amino acid sequence for function, and͞or selection pressure against aggregation. The rate-limiting step for human Pin1 WW domain folding is the formation of the loop 1 substructure. The native conformation of this six-residue loop positions side chains that are important for mediating protein-protein interactions through the binding of Pro-rich sequences. Replacement of the wild-type loop 1 primary structure by shorter sequences with a high propensity to fold into a type-I ␤-turn conformation or the statistically preferred type-I G1 bulge conformation accelerates WW domain folding by almost an order of magnitude and increases thermodynamic stability. However, loop engineering to optimize folding energetics has a significant downside: it effectively eliminates WW domain function according to ligand-binding studies. The energetic contribution of loop 1 to ligand binding appears to have evolved at the expense of fast folding and additional protein stability. Thus, the two-state barrier exhibited by the wild-type human Pin1 WW domain principally results from functional requirements, rather than from physical constraints inherent to even the most efficient loop formation process.␤-turn ͉ ligand binding ͉ protein folding ͉ ␤-sheet ͉ protein function G lobular proteins evolve by mutation and selection. Selection criteria include function, and sufficient thermodynamic stability and folding rate to avoid sustained chaperone binding and proteasome degradation. The selection criteria cannot always be optimized independently over the entire sequence of a protein. For the human Pin1 (hPin1) WW domain (Pin WW hereafter), we have shown that residues important for stability and folding rate are segregated in the sequence (1-4). It is likely that functional selection criteria are predominant once minimal energetic criteria are met. Therefore, sequence evolution to enhance function may lead to a decrease in protein stability and folding rate compared with a sequence optimized for folding energetics.The hPin1 cell cycle regulatory proline (Pro) cis͞trans-isomerase is a two-domain protein (5). In its physiological role, the N-terminal WW domain binds Pro-rich ligands of the consensus sequence (pS͞pT)P, whereas the C-terminal domain catalyzes the Pro cis͞ trans-isomerization at the pS͞pT-P peptide bond. NMR solution studies show that the two domains, which are connected by a flexible solvated linker, interact only weakly before ligand binding (6, 7). The structure of the isolated Pin WW domain is virtually superimposable on that of the WW domain in the two-domain hPin1 protein (8). Moreover, Pin WW exhibits sufficient thermodynamic stability for biophysical analysis, folds rapidly, and retains its ligand-binding function (3, 9). These attributes, combined with sequence information on Ͼ150 WW domain family members (10, 11), makes Pin WW an excellent small model p...

“…To meet these goals, we sought a single protein into which several types of enhanced aromatic sequons and their corresponding reverse turn types could be inserted without changing the overall structure or the flanking sequences. The WW domain is ideal for these requirements: Many WW variants harboring different reverse turn types in loop 1 have been structurally (34,36,37) and biophysically (36)(37)(38)(39) characterized. Crystal structures exist for WW domains harboring a type II β-turn in a six-residue loop (Fig.…”

mentioning

confidence: 99%

Glycosylation of the enhanced aromatic sequon is similarly stabilizing in three distinct reverse turn contexts

Price

Powers

et al. 2011

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146

Cotranslational N-glycosylation can accelerate protein folding, slow protein unfolding, and increase protein stability, but the molecular basis for these energetic effects is incompletely understood. N-glycosylation of proteins at naïve sites could be a useful strategy for stabilizing proteins in therapeutic and research applications, but without engineering guidelines, often results in unpredictable changes to protein energetics. We recently introduced the enhanced aromatic sequon as a family of portable structural motifs that are stabilized upon glycosylation in specific reverse turn contexts: a five-residue type I β-turn harboring a G1 β-bulge (using a Phe-Yyy-Asn-Xxx-Thr sequon) and a type II β-turn within a sixresidue loop (using a Phe-Yyy-Zzz-Asn-Xxx-Thr sequon) [Culyba EK, et al. (2011) Science 331:571-575]. Here we show that glycosylating a new enhanced aromatic sequon, Phe-Asn-Xxx-Thr, in a type I′ β-turn stabilizes the Pin 1 WW domain. Comparing the energetic effects of glycosylating these three enhanced aromatic sequons in the same host WW domain revealed that the glycosylation-mediated stabilization is greatest for the enhanced aromatic sequon complementary to the type I β-turn with a G1 β-bulge. However, the portion of the stabilization from the tripartite interaction between Phe, Asn(GlcNAc), and Thr is similar for each enhanced aromatic sequon in its respective reverse turn context. Adding the Phe-Asn-Xxx-Thr motif (in a type I′ β-turn) to the enhanced aromatic sequon family doubles the number of proteins that can be stabilized by glycosylation without having to alter the native reverse turn type.glycoprotein | biopharmaceutical | conjugate | native state stabilization | β-sheet