The structural basis for biphasic kinetics in the folding of the WW domain from a formin-binding protein: Lessons for protein design?

Abstract

The mechanism of formation of ␤-sheets is of great importance because of the significant role of such structures in the initiation and propagation of amyloid diseases. In this study we examine the folding of a series of three-stranded antiparallel ␤-sheets known as WW domains. Whereas other WW domains have been shown to fold with single-exponential kinetics, the WW domain from murine formin-binding protein 28 has recently been shown to fold with biphasic kinetics. By using a combination of kinetics and thermodynamics to characterize a simple model for this protein, the origins of the biphasic kinetics is found to lie in the fact that most of the protein is able to fold without requiring one of the ␤-hairpins to be correctly registered. The correct register of this hairpin is enforced by a surface-exposed hydrophobic contact, which is not present in other WW domains. This finding suggests the use of judiciously chosen surface-exposed hydrophobic pairs as a protein design strategy for enforcing the desired strand registry.␤-sheet ͉ ␤-strand ͉ negative design ͉ strand register A n understanding of the mechanism by which proteins reach their particular native state from the vast number of unfolded conformations will have broad-reaching implications, ranging from the prediction of protein structure from sequence to understanding diseases that originate from protein misfolding. Small model peptides and proteins have lent invaluable insight into the protein-folding process, as they afford simple systems in which the general features of folding may be elucidated (1, 2).Because of the local nature of the interactions, the formation of helices has proven considerably easier to understand using simple models (3-5) than has the formation of ␤-sheets. Accordingly, the principles that govern the formation of ␤-sheets are not well understood, despite the fact that the formation of intermolecular ␤-sheets is thought to be the crucial event in the initiation and propagation of amyloid diseases such as Alzheimer's disease (6) and spongiform encephalopathy (7).To further an understanding of the elements responsible for stability in ␤-sheets, de novo design methods have, in two cases, been used to construct three-stranded antiparallel ␤-sheets (8, 9), which have subsequently become the subject of both theoretical (10-12) and experimental (13, 14) folding studies. To ensure the generality of results toward natural proteins, however, we opt to study a series of three-stranded antiparallel ␤-sheet domains found in a variety of proteins: the WW domains (Fig. 1a).WW domains, which bind proline-rich peptide sequences, have been identified in Ͼ200 nonredundant proteins to date (15). Because of the attractiveness of WW domains as a model for ␤-sheet formation, they have been the focus of several previous folding studies. The initial study of these systems examined the thermodynamics and kinetics of folding of the human Yes-associated protein (hYAP) WW domain (16). A subsequent study made use of the more stable Pin WW domain, to characteriz...

Preventing fibril formation of a protein by selective mutation

“…For example, one of the members of the WW domain family (8, 9), the triple β-stranded WW domain from the Formin binding protein 28 (FBP28; Protein Data Bank ID code 1E0L) (10) (Fig. 1N), has been shown to fold with biphasic kinetics exhibiting intermediates during folding (3,5,6,(11)(12)(13)(14)(15)(16). We address this problem here with the design of new FBP28 WW domain mutants and by examining their structural properties and folding kinetics.…”

“…fibril formation | selective mutation | FBP28 WW domain | millisecondtimescale MD simulations | high-resolution NMR spectroscopy A n intermediate state in protein folding is involved in amyloid fibril formation, which is responsible for a number of neurodegenerative diseases (1)(2)(3)(4)(5)(6)(7). Therefore, prevention of the aggregation of folding intermediates is one of the most important problems to surmount.…”

“…Because of the small size, fast folding kinetics, and biological importance, the formation of intermolecular β-sheets is thought to be a crucial event in the initiation and propagation of amyloid diseases, such as Alzheimer's disease, and spongiform encephalopathy, FBP28, and other WW domain proteins (e.g., Pin1 and FiP35) have been the subjects of extensive experimental (4,11,(17)(18)(19)(20)(21)(22)(23) and theoretical (3,5,6,(12)(13)(14)(15)(16)(24)(25)(26)(27) studies. However, a folding mechanism of the FBP28 was debatable for a long time because of its complexity.…”

Protein folding: Then and now

“…For example, recent studies have demonstrated an agreement between theoretical and experimental folding free energy landscapes [154][155][156][157][158][159][160]. The characterization of molecular interactions responsible for different pathways opens the possibility to manipulate folding pathways.…”

Modified Replica Exchange Simulation Methods for Local Structure Refinement

“…Moreover, the transition probability is constructed such that the canonical ensemble properties are maintained during each simulation, thus providing potentially useful information about conformational probabilities as a function of temperature. Due to these advantages, REMD has been widely applied to studies of peptide and small protein folding 29,[31][32][33][34][35][36][37][38][39][40][41] .…”