Three-stranded Antiparallel Β-sheet Research Articles

The folding mechanism of a β-sheet: the WW domain

The folding thermodynamics and kinetics of the Pin WW domain, a three-stranded antiparallel β-sheet, have been characterized extensively. Folding and activation free energies were determined as a function of temperature for 16 mutants, which sample all strands and turns of the molecule. The mutational phi value (Φ m) diagram is a smooth function of sequence, indicating a prevalence of local interactions in the transition state (TS). At 37 °C, the diagram has a single pronounced maximum at turn 1: the rate-limiting step during folding is the formation of loop 1. In contrast, key residues for thermodynamic stability are located in the strand hydrophobic clusters, indicating that factors contributing to protein stability and folding kinetics are not correlated. The location of the TS along the entropic reaction coordinate Φ T, obtained by temperature-tuning the kinetics, reveals that sufficiently destabilizing mutants in loop 2 or in the Leu7-Trp11-Tyr24-Pro37 hydrophobic cluster can cause a switch to a late TS. Φ m analysis is usually applied “perturbatively” (methyl truncation), but with Φ T to quantitatively assess TS shifts along a reaction coordinate, more severe mutations can be used to probe regions of the free energy surface beyond the TS.

Role of native topology investigated by multiple unfolding simulations of four SH3 domains

The relative importance of amino acid sequence and native topology in the unfolding process of two SH3 domains and two circular permutants was investigated by 120 molecular dynamics runs at 375 K for a total simulation time of 0.72 μs. The α-spectrin (aSH3) and src SH3 (sSH3) domains, which have the same topology and a sequence identity of only 34 %, show similar unfolding pathways. The disappearance of the three-stranded antiparallel β-sheet is the last unfolding event, in agreement with a large repertoire of kinetic data derived from point mutations as well as glycine insertions and disulfide crosslinks. Two alternative routes of β-sheet unfolding have emerged from the analysis of the trajectories. One is statistically preferred in aSH3 (n-src loop breaks before distal hairpin) and the inverse in sSH3. An elongation of the β2-β3 hairpin was observed during the unfolding of sSH3 at 375 K and in 300 K simulations started from the putative transition state of sSH3 in accord with unusual kinetic data for point mutations at the n-src loop. The change of connectivity in the permutants influenced the sequence of unfolding events mainly at the permutation site. Regions where the connectivity remained unaffected showed the same chronology of contact disappearance. Taken together with previous folding simulations of two designed three-stranded antiparallel β-sheet peptides, these results indicate that, at least for small β-sheet proteins, the folding mechanism is primarily defined by the native state topology, whilst specific interactions determine the statistically predominant folding route.

Identification of Receptor Binding and Activation Determinants in the N-terminal and N-loop Regions of the CC Chemokine Eotaxin

Eotaxin is a CC chemokine that specifically activates the receptor CCR3 causing accumulation of eosinophils in allergic diseases and parasitic infections. Twelve amino acid residues in the N-terminal (residues 1-8) and N-loop (residues 11-20) regions of eotaxin have been individually mutated to alanine, and the ability of the mutants to bind and activate CCR3 has been determined in cell-based assays. The alanine mutants at positions Thr(7), Asn(12), Leu(13), and Leu(20) show near wild type binding affinity and activity. The mutants T8A, N15A, and K17A have near wild type binding affinity for CCR3 but reduced receptor activation. A third class of mutants, S4A, V5A, R16A, and I18A, display significantly perturbed binding affinity for CCR3 while retaining the ability to activate or partially activate the receptor. Finally, the mutant Phe(11) has little detectable activity and 20-fold reduced binding affinity relative to wild type eotaxin, the most dramatic effect observed in both assays but less dramatic than the effect of mutating the corresponding residue in some other chemokines. Taken together, the results indicate that residues contributing to receptor binding affinity and those required for triggering receptor activation are distributed throughout the N-terminal and N-loop regions. This conclusion is in contrast to the separation of binding and activation functions between N-loop and N-terminal regions, respectively, that has been observed previously for some other chemokines.

Native topology or specific interactions: what is more important for protein folding?

Fifty-five molecular dynamics runs of two three-stranded antiparallel β-sheet peptides were performed to investigate the relative importance of amino acid sequence and native topology. The two peptides consist of 20 residues each and have a sequence identity of 15 %. One peptide has Gly-Ser (GS) at both turns, while the other has d-Pro-Gly ( DPG). The simulations successfully reproduce the NMR solution conformations, irrespective of the starting structure. The large number of folding events sampled along the trajectories at 360 K (total simulation time of about 5 μs) yield a projection of the free-energy landscape onto two significant progress variables. The two peptides have compact denatured states, similar free-energy surfaces, and folding pathways that involve the formation of a β-hairpin followed by consolidation of the unstructured strand. For the GS peptide, there are 33 folding events that start by the formation of the 2-3 β-hairpin and 17 with first the 1-2 β-hairpin. For the DPG peptide, the statistical predominance is opposite, 16 and 47 folding events start from the 2-3 β-hairpin and the 1-2 β-hairpin, respectively. These simulation results indicate that the overall shape of the free-energy surface is defined primarily by the native-state topology, in agreement with an ever-increasing amount of experimental and theoretical evidence, while the amino acid sequence determines the statistically predominant order of the events.

Structure, Folding, and Energetics of Cooperative Interactions between the β-Strands of a de Novo Designed Three-Stranded Antiparallel β-Sheet Peptide

The effect of cooperative interactions between β-strands in enhancing β-sheet stability has been examined quantitatively by NMR using rationally designed synthetic peptides [β-hairpin (2β) and related 24-residue three-stranded antiparallel β-sheet (3β)] which are significantly folded in aqueous solution. The two hairpin components of 3β show quite different temperature-dependent stability profiles showing that a two-state model for folding (random coil ↔ three-stranded antiparallel β-sheet) is inappropriate. A four-state model for folding, involving intermediate C- and N-terminal β-hairpin conformations, is more consistent with the data. Thermodynamic analysis shows that folding of the C-terminal hairpin of 3β is entropy-driven, as previously described for the isolated hairpin 2β, but that the N-terminal hairpin, which is stabilized by a motif of aromatic residues (W4, F6, and Y11), is enthalpy-driven, consistent with stabilization through π−π interactions that are electrostatic in origin. NOE data, as we...

Mapping the transition state of the WW domain β-sheet

The folding kinetics of a three-stranded antiparallel β-sheet (WW domain) have been measured by temperature jump relaxation. Folding and activation free energies were determined as a function of temperature for both the wild-type and the mutant domain, W39F, which modifies the β 2-β 3 hydrophobic interface. The folding rate decreases at higher temperatures as a result of the increase in the activation free energy for folding. Φ-Values were obtained for thermal perturbations allowing the primary features of the folding free energy surface to be determined. The results of this analysis indicate a significant shift from an “early” (Φ T = 0.4) to a “late” (Φ T = 0.8) transition state with increasing temperature. The temperature-dependent Φ-value analysis of the wild-type WW domain and of its more stable W39F hydrophobic cluster mutant reveals little participation of residue 39 in the transition state at lower temperature. As the temperature is raised, hydrophobic interactions at the β 2-β 3 interface gain importance in the transition state and the barrier height of the wild-type, which contains the larger tryptophan residue, increases more slowly than the barrier height of the mutant.

Structure and Mechanism of 3-Deoxy-d-manno-octulosonate 8-Phosphate Synthase

3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate. KDO8P is the phosphorylated precursor of 3-deoxy-D-manno-octulosonate, an essential sugar of the lipopolysaccharide of Gram-negative bacteria. The crystal structure of the Escherichia coli KDO8P synthase has been determined by multiple wavelength anomalous diffraction and the model has been refined to 2.4 A (R-factor, 19.9%; R-free, 23.9%). KDO8P synthase is a homotetramer in which each monomer has the fold of a (beta/alpha)(8) barrel. On the basis of the features of the active site, PEP and A5P are predicted to bind with their phosphate moieties 13 A apart such that KDO8P synthesis would proceed via a linear intermediate. A reaction similar to KDO8P synthesis, the condensation of phosphoenolpyruvate, and erythrose 4-phosphate to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P), is catalyzed by DAH7P synthase. In the active site of DAH7P synthase the two substrates PEP and erythrose 4-phosphate appear to bind in a configuration similar to that proposed for PEP and A5P in the active site of KDO8P synthase. This observation suggests that KDO8P synthase and DAH7P synthase evolved from a common ancestor and that they adopt the same catalytic strategy.

Crystal structure of the surfactin synthetase-activating enzyme Sfp: a prototype of the 4'-phosphopantetheinyl transferase superfamily

The Bacillus subtilis Sfp protein activates the peptidyl carrier protein (PCP) domains of surfactin synthetase by transferring the 4'-phosphopantetheinyl moiety of coenzyme A (CoA) to a serine residue conserved in all PCPs. Its wide PCP substrate spectrum renders Sfp a biotechnologically valuable enzyme for use in combinatorial non-ribosomal peptide synthesis. The structure of the Sfp-CoA complex determined at 1.8 A resolution reveals a novel alpha/beta-fold exhibiting an unexpected intramolecular 2-fold pseudosymmetry. This suggests a similar fold and dimerization mode for the homodimeric phosphopantetheinyl transferases such as acyl carrier protein synthase. The active site of Sfp accommodates a magnesium ion, which is complexed by the CoA pyrophosphate, the side chains of three acidic amino acids and one water molecule. CoA is bound in a fashion that differs in many aspects from all known CoA-protein complex structures. The structure reveals regions likely to be involved in the interaction with the PCP substrate.

Stability of the β-Sheet of the WW Domain: A Molecular Dynamics Simulation Study

The WW domain consists of ∼40 residues, has no disulfide bridges, and forms a three-stranded antiparallel β-sheet that is monomeric in solution. It thus provides a model system for studying β-sheet stability in native proteins. We performed molecular dynamics simulations of two WW domains, YAP65 and FBP28, with very different stability characteristics, in order to explore the initial unfolding of the β-sheet. The less stable YAP domain is much more sensitive to simulation conditions than the FBP domain. Under standard simulation conditions in water (with or without charge-balancing counterions) at 300 K, the β-sheet of the YAP WW domain disintegrated at early stages of the simulations. Disintegration commenced with the breakage of a hydrogen bond between the second and third strands of the β-sheet due to an anticorrelated transition of the Tyr-28 ψ and Phe-29 ϕ angles. Electrostatic interactions play a role in this event, and the YAP WW domain structure is more stable when simulated with a complete explicit model of the surrounding ionic strength. Other factors affecting stability of the β-sheet are side-chain packing, the conformational entropy of the flexible chain termini, and the binding of cognate peptide.

NMR structure of the N-terminal domain of Saccharomyces cerevisiae RNase HI reveals a fold with a strong resemblance to the N-terminal domain of ribosomal protein L9

In addition to the conserved and well-defined RNase H domain, eukaryotic RNases HI possess either one or two copies of a small N-terminal domain. The solution structure of one of the N-terminal domains from Saccharomyces cerevisiae RNase HI, determined using NMR spectroscopy, is presented. The 46 residue motif comprises a three-stranded antiparallel β-sheet and two short α-helices which pack onto opposite faces of the β-sheet. Conserved residues involved in packing the α-helices onto the β-sheet form the hydrophobic core of the domain. Three highly conserved and solvent exposed residues are implicated in RNA binding, W22, K38 and K39. The β-β-α-β-α topology of the domain differs from the structures of known RNA binding domains such as the double-stranded RNA binding domain (dsRBD), the hnRNP K homology (KH) domain and the RNP motif. However, structural similarities exist between this domain and the N-terminal domain of ribosomal protein L9 which binds to 23 S ribosomal RNA.

Crystal structure of tobacco PR-5d protein at 1.8 Å resolution reveals a conserved acidic cleft structure in antifungal thaumatin-like proteins

The crystal structure of tobacco PR-5d, an antifungal thaumatin-like protein isolated from cultured tobacco cells, was determined at the resolution of 1.8 Å. The structure consists of 208 amino acid residues and 89 water molecules with a crystallographic R-factor of 0.169. The model has good stereochemistry, with respective root-mean-square deviations from the ideal values for bond and angle distances of 0.007 Å and 1.542 °. Of the homologous PR-5 proteins, only those with antifungal activity had a common motif, a negatively charged surface cleft. This cleft is at the boundary between domains I and II, with a bottom part consisting of a three-stranded antiparallel β-sheet in domain I. The acidic residues located in the hollow of the cleft form the β-sheet region. Sequence and secondary structure analyses showed that the amino acid residues comprising the acidic cleft of PR-5d are conserved among other antifungal PR-5 proteins. This is the first report on the high-resolution crystal structure of an antifungal PR-5 protein. This structure provides insight into the function of pathogenesis-related proteins.

Crystal structure of CcdB, a topoisomerase poison from E. coli

The crystal structure of CcdB, a protein that poisons Escherichia coli gyrase, was determined in three crystal forms. The protein consists of a five-stranded antiparallel β-pleated sheet followed by a C-terminal α-helix. In one of the loops of the sheet, a second small three-stranded antiparallel β-sheet is inserted that sticks out of the molecule as a wing. This wing contains the LysC proteolytic cleavage site that is protected by CcdA and, therefore, forms a likely CcdA recognition site. A dimer is formed by sheet extension and by extensive hydrophobic contacts involving three of the five methionine residues and the C terminus of the α-helix. The surface of the dimer on the side of the α-helix is overall negatively charged, while the opposite side as well as the wing sheet is dominated by positive charges. We propose that the CcdB dimer binds into the central hole of the 59 kDa N-terminal fragment of GyrA, after disruption of the head dimer interface of GyrA.

Structure of the IGF-binding domain of the insulin-like growth factor-binding protein-5 (IGFBP-5): implications for IGF and IGF-I receptor interactions.

Binding proteins for insulin-like growth factors (IGFs) IGF-I and IGF-II, known as IGFBPs, control the distribution, function and activity of IGFs in various cell tissues and body fluids. Insulin-like growth factor-binding protein-5 (IGFBP-5) is known to modulate the stimulatory effects of IGFs and is the major IGF-binding protein in bone tissue. We have expressed two N-terminal fragments of IGFBP-5 in Escherichia coli; the first encodes the N-terminal domain of the protein (residues 1-104) and the second, mini-IGFBP-5, comprises residues Ala40 to Ile92. We show that the entire IGFBP-5 protein contains only one high-affinity binding site for IGFs, located in mini-IGFBP-5. The solution structure of mini-IGFBP-5, determined by nuclear magnetic resonance spectroscopy, discloses a rigid, globular structure that consists of a centrally located three-stranded anti-parallel beta-sheet. Its scaffold is stabilized further by two inside packed disulfide bridges. The binding to IGFs, which is in the nanomolar range, involves conserved Leu and Val residues localized in a hydrophobic patch on the surface of the IGFBP-5 protein. Remarkably, the IGF-I receptor binding assays of IGFBP-5 showed that IGFBP-5 inhibits the binding of IGFs to the IGF-I receptor, resulting in reduction of receptor stimulation and autophosphorylation. Compared with the full-length IGFBP-5, the smaller N-terminal fragments were less efficient inhibitors of the IGF-I receptor binding of IGFs.

Molecular Models for the two Discoidin Domains of Human Blood Coagulation Factor V

Discoidin (DS) domains occur in a large variety of proteins. We have recently reported the D1 domain of galactose oxidase (GOase), a copper-containing enzyme whose structure has been determined at 1.7 A resolution, as distant member of the DS domain family. The D1 domain of GOase consists of a five-stranded antiparallel β-sheet packing against a three-stranded antiparallel β-sheet. We here show that it is possible to build 3D models for DS domains using GOase as initial template and propose a 3D structure for the C1 and C2 domains of factor V (residues 1879-2037 and 2038-2196). Factors V (FV) and VIII (FVIII) are essential and homologous non-enzymatic cofactors in the coagulation cascade. They share the domain organization A1-A2-B-A3-C1 and C2 and their C domains are members of the DS family. The C1 and C2 domains of FV are rich in positively charged residues. Several clusters of amino acids, most likely involved in inter-domain interactions, protein-protein interactions and/or phospholipid binding, are identified. Our report opens new avenues to study the structure-function relationships of DS domains.

High-resolution structure of a polyomavirus VP1-oligosaccharide complex: implications for assembly and receptor binding.

The crystal structure of a recombinant polyomavirus VP1 pentamer (residues 32-320) in complex with a branched disialylated hexasaccharide receptor fragment has been determined at 1.9 A resolution. The result extends our understanding of oligosaccharide receptor recognition. It also suggests a mechanism for enhancing the fidelity of virus assembly. We have previously described the structure of the complete polyomavirus particle complexed with this receptor fragment at 3.65 A. The model presented here offers a much more refined view of the interactions that determine carbohydrate recognition and allows us to assign additional specific contacts, in particular those involving the (alpha2,6)-linked, branching sialic acid. The structure of the unliganded VP1 pentamer, determined independently, shows that the oligosaccharide fits into a preformed groove and induces no measurable structural rearrangements. A comparison with assembled VP1 in the virus capsid reveals a rearrangement of residues 32-45 at the base of the pentamer. This segment may help prevent the formation of incorrectly assembled particles by reducing the likelihood that the C-terminal arm will fold back into its pentamer of origin.

The refined crystal structure of Bacillus cereus oligo-1,6-glucosidase at 2.0 å resolution: structural characterization of proline-substitution sites for protein thermostabilization

The crystal structure of oligo-1,6-glucosidase (dextrin 6-α-glucanohydrolase, EC 3.2.1.10) from Bacillus cereus ATCC7064 has been refined to 2.0 Å resolution with an R-factor of 19.6% for 43,328 reflections. The final model contains 4646 protein atoms and 221 ordered water molecules with respective root-mean-square deviations of 0.015 Å for bond lengths and of 3.166° for bond angles from the ideal values. The structure consists of three domains: the N-terminal domain (residues 1 to 104 and 175 to 480), the subdomain (residues 105 to 174) and the C-terminal domain (residues 481 to 558). The N-terminal domain takes a (β/α) 8-barrel structure with additions of an α-helix (Nα6′) between the sixth strand Nβ6 and the sixth helix Nα6, an α-helix (Nα7′) between the seventh strand Nβ7 and the seventh helix Nα7 and three α-helices (Nα8′, Nα8″ and Nα8‴) between the eighth strand Nβ8 and the eighth helix Nα8. The subdomain is composed of an α-helix, a three-stranded antiparallel β-sheet, and long intervening loops. The C-terminal domain has a β-barrel structure of eight antiparallel β-strands folded in double Greek key motifs, which is distorted in the sixth strand Cβ6. Three catalytic residues, Asp199, Glu255 and Asp329, are located at the bottom of a deep cleft formed by the subdomain and a cluster of the two additional α-helices Nα8′ and Nα8″ in the (β/α) 8-barrel. The refined structure reveals the locations of 21 proline-substitution sites that are expected to be critical to protein thermostabilization from a sequence comparison among three Bacillus oligo-1,6-glucosidases with different thermostability. These sites lie in loops, β-turns and α-helices, in order of frequency, except for Cys515 in the fourth β-strand Cβ4 of the C-terminal domain. The residues in β-turns (Lys121, Glu208, Pro257, Glu290, Pro443, Lys457 and Glu487) are all found at their second positions, and those in α-helices (Asn109, Glu175, Thr261 and Ile403) are present at their N1 positions of the first helical turns. Those residues in both secondary structures adopt φ and ϕ values favorable for proline substitution. Residues preceding the 21 sites are mostly conserved upon proline occurrence at these 21 sites in more thermostable Bacillus oligo-1,6-glucosidases. These structural features with respect to the 21 sites indicate that the sites in β-turns and α-helices have more essential prerequisites for proline substitution to thermostabilize the protein than those in loops. This well supports the previous finding that the replacement at the appropriate positions in β-turns or α-helices is the most effective for protein thermostabilization by proline substitution.

FBP WW domains and the Abl SH3 domain bind to a specific class of proline-rich ligands.

WW domains are conserved protein motifs of 38-40 amino acids found in a broad spectrum of proteins. They mediate protein-protein interactions by binding proline-rich modules in ligands. A 10 amino acid proline-rich portion of the morphogenic protein, formin, is bound in vitro by both the WW domain of the formin-binding protein 11 (FBP11) and the SH3 domain of Abl. To explore whether the FBP11 WW domain and Abl SH3 domain bind to similar ligands, we screened a mouse limb bud expression library for putative ligands of the FBP11 WW domain. In so doing, we identified eight ligands (WBP3 through WBP10), each of which contains a proline-rich region or regions. Peptide sequence comparisons of the ligands revealed a conserved motif of 10 amino acids that acts as a modular sequence binding the FBP11 WW domain, but not the WW domain of the putative signal transducing factor, hYAP65. Interestingly, the consensus ligand for the FBP11 WW domain contains residues that are also required for binding by the Abl SH3 domain. These findings support the notion that the FBP11 WW domain and the Abl SH3 domain can compete for the same proline-rich ligands and suggest that at least two subclasses of WW domains exist, namely those that bind a PPLP motif, and those that bind a PPXY motif.

Site-Directed Mutation Study on Hyperthermostability of Rubredoxin from Pyrococcus furiosus Using Molecular Dynamics Simulations in Solution

The hyperthermostable protein rubredoxin from Pyrococcus furiosus is a 53-residue protein with a three-stranded antiparallel β-sheet and several loops. In this paper, the hydrophobic interaction of residues on the surface of the protein was investigated as well as electrostatic interactions between residues. To investigate the effect of changes of electrostatic and hydrophobic interactions on the structure and the dynamic property of P. furiosus rebredoxin, molecular dynamics simulations in solution were performed on three mesophilic rubredoxins, P. furiosus rubredoxin, and five mutants of P. furiosus rubredoxin. Glu 14 of P. furiosus has a backbone hydrogen bond with N-terminal and multiple electrostatic interactions with Ala 1, Trp 3, and Phe 29. The multiple electrostatic interactions make the residues around the N-terminal stable, and the hydrogen bond between Glu 14 and Ala 1 remains even at high temperature. The flexibility of a loop from Asp 15 to Gly 26 is reduced by making the loop closer to the ...

Dissecting the effects of cooperativity on the stabilisation of a de novo designed three stranded anti-parallel β-sheet

Cooperative effects between two sets of weak interactions at different hydrogen bonded interfaces are shown to stabilise a de novo designed three-stranded anti-parallel β-sheet: NMR and CD (circular dichroism) spectroscopy have been used to compare and contrast the stability of the β-sheet (residues 1–24) and the isolated C-terminal β-hairpin (residues 9–24).

Tertiary Structure Comparison of Bromelain Inhibitor VI from Pineapple Stem with Bovine Pancreatic Trypsin Inhibitor.

Bromelain inhibitor VI from pineapple stem (BI-VI) is a unique double-chain cysteine proteinase inhibitor composed of an 11-residue light chain and a 41-residue heavy chain linked by disulfide bonds. The positions of six of the ten half cystines in the primary structure of BI-VI resemble those of the six half cystines in bovine pancreatic trypsin inhibitor (BPTI), a serine proteinase inhibitor which apparently shares some properties with BI-VI. These facts suggested that they might resemble in their three-dimensional structures. To clarify this point, the solution structure of BI-VI was elucidated using nuclear magnetic resonance spectroscopy and simulated annealing-based calculations. Thus, the inhibitor was shown to be composed of two distinct domains, each constructed by a three-stranded antiparallel β-sheet. Comparison with BPTI revealed conclusively that BI-VI is distinctly different in three-dimensional structure from BPTI.