The mechanism of beta-hairpin formation

Beta-hairpins constitute an important class of connecting protein secondary structures. Several groups have postulated that such structures form early in the folding process and serve to nucleate the formation of extended beta-sheet structures. Despite the importance of beta-hairpins in protein folding, little is known about the mechanism of formation of these structures. While it is well established that there is a complex interplay between the stability of a beta-hairpin and loop conformational propensity, loop length, and the formation of stabilizing cross-strand interactions (H-bonds and hydrophobic interactions), the influence of these factors on the folding rate is poorly understood. Peptide models provide a simple framework for exploring the molecular details of the formation of beta-hairpin structures. We have explored the fundamental processes of folding in two linear peptides that form beta-hairpin structures, having a stabilizing hydrophobic cluster connected by loops of differing lengths. This approach allows us to evaluate existing models of the mechanism of beta-hairpin formation. We find a substantial acceleration of the folding rate when the connecting loop is made shorter (i.e., the hydrophobic cluster is moved closer to the turn). Analysis of the folding kinetics of these two peptides reveals that this acceleration is a direct consequence of the reduced entropic cost of the smaller loop search.     

A systematic study of the vibrational free energies of polypeptides in folded and random states

Molecular vibrations, especially low frequency motions, may be used as an indication of the rigidity or the flatness of the protein folding energy landscape. We have studied the vibrational properties of native folded as well as random coil structures of more than 60 polypeptides. The picture we obtain allows us to perceive how and why the energy landscape progressively rigidifies while still allowing potential flexibility. Compared with random coil structures, both alpha-helices and beta-hairpins are vibrationally more flexible. The vibrational properties of loop structures are similar to those of the corresponding random coil structures. Inclusion of an alpha-helix tends to rigidify peptides and so-called building blocks of the structure, whereas the addition of a beta-structure has less effect. When small building blocks coalesce to form larger domains, the protein rigidifies. However, some folded native conformations are still found to be vibrationally more flexible than random coil structures, for example, beta(2)-microglobulin and the SH3 domain. Vibrational free energy contributes significantly to the thermodynamics of protein folding and affects the distribution of the conformational substates. We found a weak correlation between the vibrational folding energy and the protein size, consistent with both previous experimental estimates and theoretical partition of the heat capacity change in protein folding.     

Solution structures of a 30-residue amino-terminal domain of the carp granulin-1 protein and its amino-terminally truncated 3-30 subfragment: implications for the conformational stability of the stack of two beta-hairpins

Carp granulins are members of an emerging class of proteins with a sequence motif encoding a parallel stack of two to four beta-hairpins. The carp granulin-1 protein forms a stack of four beta-hairpins, whereas its amino-terminal fragment appears to adopt a very stable stack of two beta-hairpins in solution. Here we determined a refined three-dimensional structure of this peptide fragment to examine potential conformational changes compared with the full-length protein. The structures were calculated with both a traditional method and a fast semiautomated method using ambiguous NMR distance restraints. The resulting sets of structures are very similar and show that a well-defined stack of two beta-hairpins is retained in the peptide. Conformational rearrangements compensating the loss of the carboxy-terminal subdomain of the native protein are restricted to the carboxy-terminal end of the peptide, the turn connecting the two beta-hairpins, and the Tyr(21) and Tyr(25) aromatic side chains. Further removal of the Val(1) and Ile(2) residues, which are part of the first beta-hairpin and components of two major hydrophobic clusters in the two beta-hairpin structure, results in the loss of the first beta-hairpin. The second beta-hairpin, which is closely associated with the first, retains a similar but somewhat less stable conformation. The invariable presence of the second beta-hairpin and the dependence of its stability on the first beta-hairpin suggest that the stack of two beta-hairpins may be an evolutionary conserved and autonomous folding unit. In addition, the high conformational stability makes the stack of two beta-hairpins an attractive scaffold for the development of peptide-based drug candidates.     

Analysis of the factors that stabilize a designed two-stranded antiparallel beta-sheet

Autonomously folding beta-hairpins (two-strand antiparallel beta-sheets) have become increasingly valuable tools for probing the forces that control peptide and protein conformational preferences. We examine the effects of variations in sequence and solvent on the stability of a previously designed 12-residue peptide (1). This peptide adopts a beta-hairpin conformation containing a two-residue loop (D-Pro-Gly) and a four-residue interstrand sidechain cluster that is observed in the natural protein GB1. We show that the conformational propensity of the loop segment plays an important role in beta-hairpin stability by comparing 1 with (D)P--> N mutant 2. In addition, we show that the sidechain cluster contributes both to conformational stability and to folding cooperativity by comparing 1 with mutant 3, in which two of the four cluster residues have been changed to serine. Thermodynamic analysis suggests that the high loop-forming propensity of the (D)PG segment decreases the entropic cost of beta-hairpin formation relative to the more flexible NG segment, but that the conformational rigidity of (D)PG may prevent optimal contacts between the sidechains of the GB1-derived cluster. The enthalpic favorability of folding in these designed beta-hairpins suggests that they are excellent scaffolds for studying the fundamental mechanisms by which amino acid sidechains interact with one another in folded proteins.     

Constraining local structure can speed up folding by promoting structural polarization of the folding pathway

The pathway which proteins take to fold can be influenced from the earliest events of structure formation. In this light, it was both predicted and confirmed that increasing the stiffness of a beta hairpin turn decreased the size of the transition state ensemble (TSE), while increasing the folding rate. Thus, there appears to be a relationship between conformationally restricting the TSE and increasing the folding rate, at least for beta hairpin turns. In this study, we hypothesize that the enormous sampling necessary to fold even two-state folding proteins in silico could be reduced if local structure constraints were used to restrict structural heterogeneity by polarizing folding pathways or forcing folding into preferred routes. Using a Gō model, we fold Chymotrypsin Inhibitor 2 (CI-2) and the src SH3 domain after constraining local sequence windows to their native structure by rigid body dynamics (RBD). Trajectories were monitored for any changes to the folding pathway and differences in the kinetics compared with unconstrained simulations. Constraining local structure decreases folding time two-fold for 41% of src SH3 windows and 45% of CI-2 windows. For both proteins, folding times are never significantly increased after constraining any window. Structural polarization of the folding pathway appears to explain these rate increases. Folding rate enhancements are consistent with the goal to reduce sampling time necessary to reach native structures during folding simulations. As anticipated, not all constrained windows showed an equal decrease in folding time. We conclude by analyzing these differences and explain why RBD may be the preferred way to constrain structure.     

Cross-strand side-chain interactions versus turn conformation in beta-hairpins.

A series of designed peptides has been analyzed by 1H-NMR spectroscopy in order to investigate the influence of cross-strand side-chain interactions in beta-hairpin formation. The peptides differ in the N-terminal residues of a previously designed linear decapeptide that folds in aqueous solution into two interconverting beta-hairpin conformations, one with a type I turn (beta-hairpin 4:4) and the other with a type I + G1 beta-bulge turn (beta-hairpin 3:5). Analysis of the conformational behavior of the peptides studied here demonstrates three favorable and two unfavorable cross-strand side-chain interactions for beta-hairpin formation. These results are in agreement with statistical data on side-chain interactions in protein beta-sheets. All the peptides in this study form significant populations of the beta-hairpin 3:5, but only some of them also adopt the beta-hairpin 4:4. The formation of beta-hairpin 4:4 requires the presence of at least two favorable cross-strand interactions, whereas beta-hairpin 3:5 seems to be less susceptible to side-chain interactions. A protein database analysis of beta-hairpins 3:5 and beta-hairpins 4:4 indicates that the former occur more frequently than the latter. In both peptides and proteins, beta-hairpins 3:5 have a larger right-handed twist than beta-hairpins 4:4, so that a factor contributing to the higher stability of beta-hairpin 3:5 relative to beta-hairpin 4:4 is due to an appropriate backbone conformation of the type I + G1 beta-bulge turn toward the right-handed twist usually observed in protein beta-sheets. In contrast, as suggested previously, backbone geometry of the type I turn is not adequate for the right-handed twist. Because analysis of buried hydrophobic surface areas on protein beta-hairpins reveals that beta-hairpins 3:5 bury more hydrophobic surface area than beta-hairpins 4:4, we suggest that the right-handed twist observed in beta-hairpin 3:5 allows a better packing of side chains and that this may also contribute to its higher intrinsic stability.     

Beta-bulges within loops as recurring features of protein structure

Beta-bulges have been described as common irregularities in antiparallel beta-sheets. The two commonest kinds are described as 'classic' and 'G1'. The G1-type is often observed to occur at the loop end of beta-hairpins. I confirm this but also find that the characteristic G1 beta-bulge features (hydrogen bonds and dihedral angles) are found, in some loops, in the absence of any adjacent antiparallel beta-hairpin, so that the beta-bulge is effectively acting as a new sort of turn. Also I show that G1 beta-bulge loops occur in a number of different structural forms, some of which have not been described in detail before. G1 beta-bulge loops are common features; on average, a half of all proteins have one.     

Crystal structure of the SH3 domain in human Fyn; comparison of the three-dimensional structures of SH3 domains in tyrosine kinases and spectrin.

The Src-homology 3 (SH3) region is a protein domain consisting of approximately 60 residues. It occurs in a large number of eukaryotic proteins involved in signal transduction, cell polarization and membrane--cytoskeleton interactions. The function is unknown, but it is probably involved in specific protein--protein interactions. Here we report the crystal structure of the SH3 domain of Fyn (a Src family tyrosine kinase) at 1.9 A resolution. The crystals have two SH3 molecules per asymmetric unit. These two Fyn SH3 domains are not related by a local twofold axis. The crystal structures of spectrin and Fyn SH3 domains as well as the solution structure of the Src SH3 domain show that these all have the same basic fold. A protein domain which has the same topology as SH3 is present in the prokaryotic regulatory enzyme BirA. The comparison between the crystal structures of Fyn and spectrin SH3 domains shows that a conserved surface patch, consisting mainly of aromatic residues, is flanked by two hairpin-like loops (residues 94-104 and 114-118 in Fyn). These loops are different in tyrosine kinase and spectrin SH3 domains. They could modulate the binding properties of the aromatic surface.     

Hierarchy of structure loss in MD simulations of src SH3 domain unfolding.

To complement experimental studies of the src SH3 domain folding, we studied 30 independent, high-temperature, molecular dynamics simulations of src SH3 domain unfolding. These trajectories were observed to differ widely from each other. Thus, rather than analyzing individual trajectories, we sought to identify the recurrent features of the high-temperature unfolding process. The conformations from all simulations were combined and then divided into groups based on the number of native contacts. Average occupancies of each side-chain hydrophobic contact and hydrogen bond in the protein were then determined. In the symmetric funnel limit, the occupancies of all contacts should decrease in concert with the loss in total number of native contacts. If there is a lack of symmetry or hierarchy to the unfolding process, the occupancies of some contacts should decrease more slowly, and others more rapidly. Despite the heterogeneity of the individual trajectories, the ensemble averaging revealed an order to the unfolding process: contacts between the N and C-terminal strands are the first to disappear, whereas contacts within the distal beta-hairpin and a hydrogen-bonding network involving the distal loop beta-turn and the diverging turn persist well after the majority of the native contacts are lost. This hierarchy of events resembles but is somewhat less pronounced than that observed in our experimental studies of the folding of src SH3 domain.     

Gating dynamics of the acetylcholine receptor extracellular domain

We used single-channel recording and model-based kinetic analyses to quantify the effects of mutations in the extracellular domain (ECD) of the alpha-subunit of mouse muscle-type acetylcholine receptors (AChRs). The crystal structure of an acetylcholine binding protein (AChBP) suggests that the ECD is comprised of a beta-sandwich core that is surrounded by loops. Here we focus on loops 2 and 7, which lie at the interface of the AChR extracellular and transmembrane domains. Side chain substitutions in these loops primarily affect channel gating by either decreasing or increasing the gating equilibrium constant. Many of the mutations to the beta-core prevent the expression of functional AChRs, but of the mutants that did express almost all had wild-type behavior. Rate-equilibrium free energy relationship analyses reveal the presence of two contiguous, distinct synchronously-gating domains in the alpha-subunit ECD that move sequentially during the AChR gating reaction. The transmitter-binding site/loop 5 domain moves first (Phi = 0.93) and is followed by the loop 2/loop 7 domain (Phi = 0.80). These movements precede that of the extracellular linker (Phi = 0.69). We hypothesize that AChR gating occurs as the stepwise movements of such domains that link the low-to-high affinity conformational change in the TBS with the low-to-high conductance conformational change in the pore.     

Bergerac-SH3: "frustation" induced by stabilizing the folding nucleus

The influence of an inserted exogenous independent folding element on the thermodynamics and folding properties of SH3 domain from alpha-spectrin has been investigated by creating a fused form between this small all-beta domain and a stable beta-hairpin (BH19). NMR analysis of synthetic peptides shows that insertion of BH19 nucleates formation of the original natural beta-hairpin (distal loop) that is part of the SH3 folding nucleus. The resulting protein (Bergerac-SHH) is more stable, folds faster and contains an elongated hairpin protruding from the globular domain as determined by 2D-NMR. "Protein engineering" analysis of the inserted region shows that it is folded in the transition state. Interestingly, stabilisation by insertion of the distal loop region results in the appearance of a compact intermediate revealed by a curved chevron plot at low denaturant concentration. This effect is eliminated at low salt concentrations by a single mutation of a hydrophobic residue within BH19 sequence, which is most probably involved in non-native interactions. Local stabilisation by enlargement and reinforcement of the folding nucleus, global compaction by the addition of salt and non-native interactions are shown to contribute to the observed deviation from the two-state behaviour.     

Specificity determinants of a novel Nck interaction with the juxtamembrane domain of the epidermal growth factor receptor

Nck is a ubiquitously expressed adaptor protein containing Src homology 2 (SH2) and Src homology 3 (SH3) domains. It integrates downstream effector proteins with cell membrane receptors, such as the epidermal growth factor receptor (EGFR). EGFR plays a critical role in cellular proliferation and differentiation. The 45-residue juxtamembrane domain of EGFR (JM), located between the transmembrane and kinase domains, regulates receptor activation and trafficking to the basolateral membrane of polarized epithelia through a proline-rich motif that resembles a consensus SH3 domain binding site. We demonstrate here that the JM region can bind to Nck, showing a notable binding preference for the second SH3 domain. To elucidate the structural determinants for this interaction, we have determined the NMR solution structures of both the first and second Nck SH3 domains (Nck1-1 and Nck1-2). These domains adopt a canonical SH3 beta-barrel-like fold, containing five antiparallel strands separated by three loop regions and one 3 10-helical turn. Chemical shift perturbation studies have identified the residues that form the binding cleft of Nck1-2, which are primarily located in the RT and n-Src loops. JM binds to Nck1-2 with an affinity of approximately 80 microM through a positively charged sequence near the N-terminus, as opposed to the polyproline sequence. The two Nck SH3 domains exhibit both steric and electrostatic differences in their RT-Src and n-Src loops, and a model of the Nck1-2 domain complexed with the JM highlights the factors that define the putative binding mode for this ligand.     

The folding transition state between SH3 domains is conformationally restricted and evolutionarily conserved.

The protein engineering analysis of the alpha-spectrin SH3 domain at three different stability conditions (pH 7.0, 3.5 and 2.5) reveals a folding transition state structured around the distal loop beta-hairpin and the 310-helix. This region is impervious to overall changes in protein stability, suggesting a transition state ensemble with little conformational variability. Comparison with the Src SH3 domain (36% sequence homology) indicates that the transition state in this protein family may be conserved. Discrepancies at some positions can be rationalized in terms of the different interactions made by the different side chains in both domains. Br?nsted plot analysis confirms the straight phi(doubledagger-U) results and shows two folding subdomains for this small protein. These results, together with previous data on circular permutants of the alpha-spectrin SH3 domain, indicate that polypeptide topology and chain connectivity play a major role in the folding reaction of this protein family.     

PhosphoThr peptide binding globally rigidifies much of the FHA domain from Arabidopsis receptor kinase-associated protein phosphatase

A net increase in the backbone rigidity of the kinase-interacting FHA domain (KI-FHA) from the Arabidopsis receptor kinase-associated protein phosphatase (KAPP) accompanies the binding of a phosphoThr peptide from its CLV1 receptor-like kinase partner, according to (15)N NMR relaxation at 11.7 and 14.1 T. All of the loops of free KI-FHA display evidence of nanosecond-scale motions. Many of these same residues have residual dipolar couplings that deviate from structural predictions. Binding of the CLV1 pT868 peptide seems to reduce nanosecond-scale fluctuations of all loops, including half of the residues of recognition loops. Residues important for affinity are found to be rigid, i.e., conserved residues and residues of the subsite for the key pT+3 peptide position. This behavior parallels SH2 and PTB domain recognition of pTyr peptides. PhosphoThr peptide binding increases KI-FHA backbone rigidity (S(2)) of three recognition loops, a loop nearby, seven strands from the beta-sandwich, and a distal loop. Compensating the trend of increased rigidity, binding enhances fast mobility at a few sites in four loops on the periphery of the recognition surface and in two loops on the far side of the beta-sandwich. Line broadening evidence of microsecond- to millisecond-scale fluctuations occurs across the six-stranded beta-sheet and nearby edges of the beta-sandwich; this forms a network connected by packing of interior side chains and H-bonding. A patch of the slowly fluctuating residues coincides with the site of segment-swapped dimerization in crystals of the FHA domain of human Chfr. Phosphopeptide binding introduces microsecond- to millisecond-scale fluctuations to more residues of the long 8/9 recognition loop of KI-FHA. The rigidity of this FHA domain appears to couple as a whole to pThr peptide binding.     

Directed discovery of bivalent peptide ligands to an SH3 domain

The Caenorhabditis elegans SEM-5 SH3 domains recognize proline-rich peptide segments with modest affinity. We developed a bivalent peptide ligand that contains a naturally occurring proline-rich binding sequence, tethered by a glycine linker to a disulfide-closed loop segment containing six variable residues. The glycine linker allows the loop segment to explore regions of greatest diversity in sequence and structure of the SH3 domain: the RT and n-Src loops. The bivalent ligand was optimized using phage display, leading to a peptide (PP-G(4)-L) with 1000-fold increased affinity for the SEM-5 C-terminal SH3 domain over that of a natural ligand. NMR analysis of the complex confirms that the peptide loop segment is targeted to the RT and n-Src loops and parts of the beta-sheet scaffold of this SH3 domain. This binding region is comparable to that targeted by a natural non-PXXP peptide to the p67(phox) SH3 domain, a region not known to be targeted in the Grb2 SH3 domain family. PP-G(4)-L may aid in the discovery of additional binding partners of Grb2 family SH3 domains.     

Folding specificity induced by loop stiffness

To test the importance of loop stiffness in restricting the heterogeneity of transition state ensemble, we relaxed the distal loop of 10 unstable redesigned hydrophobic core mutants of alpha-spectrin SH3 domain. This was achieved by replacing Asp48 by Gly at the tip of the distal hairpin. Although the change was local in nature, the effect on stabilization was not uniform across the core mutants tested. There is an inverse rough correlation between the stabilization and the increase in buried hydrophobic volume, with respect to the wild type. Interestingly enough, proteins that although unstable are properly folded become molten globule-like after relaxation of the distal loop. These results highlight the importance of stiffness in restricting the conformational heterogeneity of a protein during the folding reaction. An interplay between unspecific hydrophobic interactions and constraint induced by polar interactions, or in this case local stiffness, is essential to achieve a well-ordered folded structure.     

Effect of salt-dependent stiffness on the conformation of a stressed DNA loop containing initially coplanar bends

Closed DNA loops containing one or more bent regions are important structures that occur in the regulation of gene expression. We analyze the response of structures of this type to a change in applied rotation (change in linking deficiency, delta Lk). Our results apply to a closed loop formed from an elastic rod that is intrinsically bent in Nb discrete, 20 degrees steps up to a maximum of 240 degrees, the bent regions being initially coplanar with the plane of the relaxed DNA loop. We determine the effect of changing the intrinsic elastic resistance of the DNA loop to bending and torsional deformations. This relative resistance is expressed by Poisson's ratio v, which depends upon the ratio of bending stiffness to torsional rigidity. Poisson's ratio is primarily a function of salt type and concentration. We find that the tertiary structure of DNA loops changes with delta Lk, but that the geometric response can be either of two quite different types, depending upon the precise (Nb, v) pair. For combinations of Nb and v that are above a critical curve (the Fickel curve), the response to increasing delta Lk is nonmonotonic (NMT region): the distance between the loop closure point and its diametric opposite first decreases, then increases, as delta Lk increases. For combinations of Nb and v that are below the Fickel curve (NMT region), the corresponding diameter never increases, but always decreases with increasing delta Lk. In addition to these results, we define and implement a new measure of tertiary structure in closed DNA: the absolute writhe, AWr.     

Structure and assembly of designed beta-hairpin peptides in crystals as models for beta-sheet aggregation

De novo designed beta-hairpin peptides have generally been recalcitrant to crystallization. The crystal structures of four synthetic peptide beta-hairpins, Boc-Leu-Val-Val-DPro-Gly-Leu-Phe-Val-OMe (1), Boc-Leu-Phe-Val-DPro-Ala-Leu-Phe-Val-OMe (2), Boc-Leu-Val-Val-DPro-Aib-Leu-Val-Val-OMe (3), and Boc-Met-Leu-Phe-Val-DPro-Ala-Leu-Val-Val-Phe-OMe (4), are described. The centrally positioned DPro-Xxx segment promotes prime beta-turn formation, thereby nucleating beta-hairpin structures. In all four peptides well-defined beta-hairpins nucleated by central type II' DPro-Xxx beta-turns have been characterized by X-ray diffraction, providing a view of eight crystallographically independent hairpins. In peptides 1-3 three intramolecular cross-strand hydrogen bonds stabilized the observed beta-hairpin, with some fraying of the structures at the termini. In peptide 4, four intramolecular cross-strand hydrogen bonds stabilized the hairpin. Peptides 1-4 reveal common features of packing of beta-hairpins into crystals. Two-dimensional sheet formation mediated by intermolecular hydrogen bonds formed between antiparallel strands of adjacent molecule is a recurrent theme. The packing of two-dimensional sheets into the crystals is mediated in the third dimension by bridging solvents and interactions of projecting side chains, which are oriented on either face of the sheet. In all cases, solvation of the central DPro-Xxx peptide unit beta-turn is observed. The hairpins formed in the octapeptides are significantly buckled as compared to the larger hairpin in peptide 4, which is much flatter. The crystal structures provide insights into the possible modes of beta-sheet packing in regular crystalline arrays, which may provide a starting point for understanding beta-sandwich and cross-beta-structures in amyloid fibrils.     

Conformation of beta-hairpins in protein structures. A systematic classification with applications to modelling by homology, electron density fitting and protein engineering

A systematic classification of beta-hairpin structures which takes into account the polypeptide chain length and hydrogen bonding between the two antiparallel beta-strands is described. We have used this classification of beta-hairpin structures and their specific sequence pattern to derive rules which demonstrate its usefulness in assisting modelling beta-hairpins. These rules can be applied to comparative model building, modelling into electron density and in the prediction of conformation of beta-hairpins to aid protein engineering.     

Protein folding kinetics beyond the phi value: using multiple amino acid substitutions to investigate the structure of the SH3 domain folding transition state

The SH3 domain folding transition state structure contains two well-ordered turn regions, known as the diverging turn and the distal loop. In the Src SH3 domain transition state, these regions are stabilized by a hydrogen bond between Glu30 in the diverging turn and Ser47 in the distal loop. We have examined the effects on folding kinetics of amino acid substitutions at the homologous positions (Glu24 and Ser41) in the Fyn SH3 domain. In contrast to most other folding kinetics studies which have focused primarily on non-disruptive substitutions with Ala or Gly, here we have examined the effects of substitutions with diverse amino acid residues. Using this approach, we demonstrate that the transition state structure is generally tolerant to amino acid substitutions. We also uncover a unique role for Ser at position 41 in facilitating folding of the distal loop, which can only be replicated by Asp at the same position. Both these residues appear to accelerate folding through the formation of short-range side-chain to backbone hydrogen bonds. The folding of the diverging turn region is shown to be driven primarily by local interactions. The diverging turn and distal loop regions are found to interact in the transition state structure, but only in the context of particular mutant backgrounds. This work demonstrates that studying the effects of a variety of amino acid substitutions on protein folding kinetics can provide unique insights into folding mechanisms which cannot be obtained by standard Phi value analysis.