Dramatic stabilization of an SH3 domain by a single substitution: roles of the folded and unfolded states

The N-terminal SH3 domain of the Drosophila drk protein (drkN SH3) exists in equilibrium between folded and unfolded states under non-denaturing buffer conditions. In order to examine the origins of this instability, we have made mutations in the domain and characterized the thermodynamics and kinetics of folding. Results of substitutions of negatively charged residues to neutral amino acid residues suggest that the large electrostatic potential of the domain does not play a dominant role in the instability of the domain. Sequence alignment of a large number of SH3 domains reveals that the drkN SH3 domain has a threonine (T22) at a position corresponding to an otherwise highly conserved glycine residue in the diverging beta-turn connecting the beta3 and beta4 strands. Mutation of T22 to glycine results in significant stabilization of the drkN SH3 domain by 2.5 kcal/mole. To further characterize the basis for the stabilization of the T22 mutant relative to wild-type, we made additional mutant proteins with substitutions of residue T22. A strong correlation is seen between protein stability or folding rate and propensity for native beta-turn structure at this position. Correlation of folding rates with AGADIR predictions of non-native helical structure in the diverging turn region, along with our previous NMR evidence for non-native structure in this region of the unfolded state of the drkN SH3 domain, suggests that the free energy of the unfolded state also plays a role in stability. This result highlights the importance of both folded and unfolded states for understanding protein stability.     

Energetics of the native energy landscape of a two-domain calcium sensor protein: distinct folding features of the two domains

The protein folding energy landscape allows a thorough understanding of the protein folding problem which in turn helps in understanding various aspects of biological functions. Characterizing the cooperative unfolding units and the intermediates along the folding funnel of a protein is a challenging task. In this paper, we investigated the native energy landscape of EhCaBP, a calcium sensor, belonging to the same EF-hand superfamily as calmodulin. EhCaBP is a two-domain EF-hand protein consisting of two EF-hands in each domain and binding to four Ca2+ cations. Native-state hydrogen exchange (HX) was used to assess the folding features of the landscape and also to throw light on the structure-folding function paradigm of calcium sensor proteins. HX measurements under the EX2 regime provided the thermodynamic information about the protein folding events under native conditions. HX studies revealed that the unfolding of EhCaBP is not a two-state process. Instead, it proceeds through cooperative units. The C-terminal domain exhibits less denaturant dependence than the N-terminal domain, suggesting that the former is dominated by local fluctuations. It is interesting to note that the N- and C-terminal domains of EhCaBP have distinct folding features. In fact, these observed differences can regulate the domain-dependent target recognition of two-domain Ca2+ sensor proteins.     

Tyrosine phosphorylation in the SH3 domain disrupts negative regulatory interactions within the c-Abl kinase core

Recent studies have shown that trans-phosphorylation of the Abl SH3 domain at Tyr89 by Src-family kinases is required for the full transforming activity of Bcr-Abl. Tyr89 localizes to a binding surface of the SH3 domain that engages the SH2-kinase linker in the crystal structure of the c-Abl core. Displacement of SH3 from the linker is likely to influence efficient downregulation of c-Abl. Hydrogen-deuterium exchange (HX) and mass spectrometry (MS) were used to investigate whether Tyr89 phosphorylation affects the ability of the SH3 domain to interact intramolecularly with the SH2-kinase linker in cis as well as other peptide ligands in trans. HX MS analysis of SH3 binding showed that when various Abl constructs were phosphorylated at Tyr89 by the Src-family kinase Hck, SH3 was unable to engage a high-affinity ligand in trans and that interaction with the linker in cis was reduced dramatically in a construct containing the SH3 and SH2 domains plus the linker. Phosphorylation of the Abl SH3 domain on Tyr89 also interfered with binding to the negative regulatory protein Abi-1 in trans. Site-directed mutagenesis of Tyr89 and Tyr245, another tyrosine phosphorylation site located in the linker that may also influence SH3 binding, implicated Tyr89 as the key residue necessary for disrupting regulation after phosphorylation. These results imply that phosphorylation at Tyr89 by Src-family kinases prevents engagement of the Abl SH3 domain with its intramolecular binding partner leading to enhanced Abl kinase activity and cellular signaling.     

Circularization changes the folding transition state of the src SH3 domain

Native state topology has been implicated as a major determinant of protein-folding mechanisms. Here, we test experimentally the robustness of the src SH3-domain folding transition state to changes in topology by covalently constraining regions of the protein with disulfide crosslinks and then performing kinetic analysis on point mutations in the context of these modified proteins. Circularization (crosslinking the N and C termini) of the src SH3 domain makes the protein topologically symmetric and causes delocalization of structure in the transition state ensemble suggesting a change in the folding mechanism. In contrast, crosslinking a single structural element (the distal beta-hairpin) which is an essential part of the transition state, results in a protein that folds 30 times faster, but does not change the distribution of structure in the transition state. As the transition states of distantly related SH3 domains were previously found to be very similar, we conclude that the free energy landscape of this protein family contains deep features which are relatively insensitive to sequence variations but can be altered by changes in topology.     

Thermodynamic and kinetic exploration of the energy landscape of Borrelia burgdorferi OspA by native-state hydrogen exchange

We report a native-state hydrogen-exchange (HX) method to simultaneously obtain both thermodynamic and kinetic information on the formation of multiple excited states in a folding energy landscape. Our method exploits the inherent dispersion and pH dependence of the intrinsic HX rates to cover both the EX2 (thermodynamic) and EX1 (kinetic) regimes. At each concentration of denaturant, HX measurements are performed over a range of pH values. Using this strategy, we dissected Borrelia burgdorferi OspA, a predominantly beta-sheet protein containing a unique single-layer beta-sheet, into five cooperative units and postulated excited states predominantly responsible for HX. More importantly, we determined the interconversion rates between these excited states and the native state. The use of both thermodynamic and kinetic information from native-state HX enabled us to construct a folding landscape of this 28kDa protein, including local minima and maxima, and to discriminate on-pathway and off-pathway intermediates. This method, which we term EX2/EX1 HX, should be a powerful tool for characterizing the complex folding mechanisms exhibited by the majority of proteins.     

The native state conformational ensemble of the SH3 domain from alpha-spectrin.

The folding/unfolding equilibrium of the alpha-spectrin SH3 domain has been measured by NMR-detected hydrogen/deuterium exchange and by differential scanning calorimetry. Protection factors against exchange have been obtained under native conditions for more than half of the residues in the domain. Most protected residues are located at the beta-strands, the short 3(10) helix, and part of the long RT loop, whereas the loops connecting secondary structure elements show no measurable protection. Apparent stability constants per residue and their corresponding Gibbs energies have been calculated from the exchange experiments. The most stable region of the SH3 domain is defined by the central portions of the beta-strands. The peptide binding region, on the other hand, is composed of a highly stable region (residues 53-57) and a highly unstable region, the loop between residues 34-41 (n-Src loop). All residues in the domain have apparent Gibbs energies lower than the global unfolding Gibbs energy measured by differential scanning calorimetry, indicating that under our experimental conditions the amide exchange of all residues in the SH3 domain occurs primarily via local unfolding reactions. A structure-based thermodynamic analysis has allowed us to predict correctly the thermodynamics of the global unfolding of the domain and to define the ensemble of conformational states that quantitatively accounts for the observed pattern of hydrogen exchange protection. These results demonstrate that under native conditions the SH3 domain needs to be considered as an ensemble of conformations and that the hydrogen exchange data obtained under those conditions cannot be interpreted by a two-state equilibrium. The observation that specific regions of a protein are able to undergo independent local folding/unfolding reactions indicates that under native conditions the scale of cooperative interactions is regional rather than global.     

The high-resolution NMR structure of a single-chain chimeric protein mimicking a SH3-peptide complex

Here we present the high-resolution NMR structure of a chimera (SPCp41) between alpha-spectrin SH3 domain and the decapeptide p41. The tertiary structure mimics perfectly the interactions typically found in SH3-peptide complexes and is remarkably similar to that of the complex between the separate Spc-SH3 domain and ligand p41. Relaxation data confirm the tight binding between the ligand and SH3 part of the chimera. This chimera will serve as a tool for a deeper understanding of the relationship between structure and thermodynamics of binding using a combination of NMR, stability and site-directed mutagenesis studies, which can lead to an effective strategy for ligand design.     

Signal Peptide-rheostat Dynamics Delay Secretory Preprotein Folding

Sec secretory proteins are distinguished from cytoplasmic ones by N-terminal signal peptides with multiple roles during post-translational translocation. They contribute to preprotein targeting to the translocase by slowing down folding, binding receptors and triggering secretion. While signal peptides get cleaved after translocation, mature domains traffic further and/or fold into functional states. How signal peptides delay folding temporarily, to keep mature domains translocation-competent, remains unclear. We previously reported that the foldon landscape of the periplasmic prolyl-peptidyl isomerase is altered by its signal peptide and mature domain features. Here, we reveal that the dynamics of signal peptides and mature domains crosstalk. This involves the signal peptide’s hydrophobic helical core, the short unstructured connector to the mature domain and the flexible rheostat at the mature domain N-terminus. Through this cis mechanism the signal peptide delays the formation of early initial foldons thus altering their hierarchy and delaying mature domain folding. We propose that sequence elements outside a protein’s native core exploit their structural dynamics to influence the folding landscape.     

A single mutation in an SH3 domain increases amyloid aggregation by accelerating nucleation, but not by destabilizing thermodynamically the native state

We investigated the relationship between thermodynamic stability and amyloid aggregation propensity for a set of single mutants of the alpha-spectrin SH3 domain (Spc-SH3). Whilst mutations destabilizing the domain at position 56 did not enhance fibrillation, the N47A mutation increased the rate of amyloid fibril formation by 10-fold. Even under conditions of identical thermodynamic stability, the aggregation rate was much higher for the N47A mutant than for the WT domain. We conclude that the N47A mutation does not change the apparent mechanism of fibrillation or the morphology of the amyloid fibrils, and that its amyloidogenic property is due to its effect upon the rate of the conformational events leading to nucleation and not to its overall destabilizing effect.     

pH dependence of the hydrogen exchange in the SH3 domain of alpha-spectrin

Using nuclear magnetic resonance we have measured the hydrogen exchange (HX) in the Src homology region 3 (SH3) domain of alpha-spectrin as a function of pH*. At very acidic pH* values the exchange of most residues appears to occur via global unfolding, although several residues show abnormally large Gibbs energies of exchange, suggesting the presence of some residual structure in the unfolded state. At higher pH* HX occurs mainly via local or partial unfoldings. We have been able to characterize the coupling between the electrostatic interactions in this domain and the conformational fluctuations occurring under native conditions by analyzing the dependence upon pH* of the Gibbs energy of exchange. The SH3 domain seems to be composed of a central core, which requires large structural disruptions to become exposed to the solvent, surrounded by smaller subdomains, which fluctuate independently.     

Predicting repeat protein folding kinetics from an experimentally determined folding energy landscape

The Notch ankyrin domain is a repeat protein whose folding has been characterized through equilibrium and kinetic measurements. In previous work, equilibrium folding free energies of truncated constructs were used to generate an experimentally determined folding energy landscape (Mello and Barrick, Proc Natl Acad Sci USA 2004;101:14102–14107). Here, this folding energy landscape is used to parameterize a kinetic model in which local transition probabilities between partly folded states are based on energy values from the landscape. The landscape‐based model correctly predicts highly diverse experimentally determined folding kinetics of the Notch ankyrin domain and sequence variants. These predictions include monophasic folding and biphasic unfolding, curvature in the unfolding limb of the chevron plot, population of a transient unfolding intermediate, relative folding rates of 19 variants spanning three orders of magnitude, and a change in the folding pathway that results from C‐terminal stabilization. These findings indicate that the folding pathway(s) of the Notch ankyrin domain are thermodynamically selected: the primary determinants of kinetic behavior can be simply deduced from the local stability of individual repeats.     

The design of a hyperstable mutant of the Abp1p SH3 domain by sequence alignment analysis

We have characterized the thermodynamic stability of the SH3 domain from the Saccharomyces cerevisiae Abp1p protein and found it to be relatively low compared to most other SH3 domains, with a Tm of 60 degrees C and a deltaGu of 3.08 kcal/mol. Analysis of a large alignment of SH3 domains led to the identification of atypical residues at eight positions in the wild-type Abp1p SH3 domain sequence that were subsequently replaced by the residue seen most frequently at that position in the alignment. Three of the eight mutants constructed in this way displayed increases in Tm ranging from 8 to 15 degrees C with concomitant increases in deltaGu of up to 1.4 kcal/mol. The effects of these substitutions on folding thermodynamics and kinetics were entirely additive, and a mutant containing all three was dramatically stabilized with a Tm greater than 90 degrees C and a deltaGu more than double that of the wild-type domain. The folding rate of this hyperstable mutant was 10-fold faster than wild-type, while its unfolding rate was fivefold slower. All of the stabilized mutants were still able to bind a target peptide with wild-type affinity. We have analyzed the stabilizing amino acid substitutions isolated in this study and several other similar sequence alignment based studies. In approximately 25% of cases, increased stability can be explained by enhanced propensity of the substituted residue for the local backbone conformation at the mutagenized site.     

Stability and peptide binding specificity of Btk SH2 domain: molecular basis for X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is caused by mutations in the Bruton's tyrosine kinase (Btk). The absence of functional Btk leads to failure of B-cell development that incapacitates antibody production in XLA patients leading to recurrent bacterial infections. Btk SH2 domain is essential for phospholipase C-gamma phosphorylation, and mutations in this domain were shown to cause XLA. Recently, the B-cell linker protein (BLNK) was found to interact with the SH2 domain of Btk, and this association is required for the activation of phospholipase C-gamma. However, the molecular basis for the interaction between the Btk SH2 domain and BLNK and the cause of XLA remain unclear. To understand the role of Btk in B-cell development, we have determined the stability and peptide binding affinity of the Btk SH2 domain. Our results indicate that both the structure and stability of Btk SH2 domain closely resemble with other SH2 domains, and it binds with phosphopeptides in the order pYEEI > pYDEP > pYMEM > pYLDL > pYIIP. We expressed the R288Q, R288W, L295P, R307G, R307T, Y334S, Y361C, L369F, and 1370M mutants of the Btk SH2 domain identified from XLA patients and measured their binding affinity with the phosphopeptides. Our studies revealed that mutation of R288 and R307 located in the phosphotyrosine binding site resulted in a more than 200-fold decrease in the peptide binding compared to L295, Y334, Y361, L369, and 1370 mutations in the pY + 3 hydrophobic binding pocket (approximately 3- to 17-folds). Furthermore, mutation of the Tyr residue at the betaD5 position reverses the binding order of Btk SH2 domain to pYIIP > pYLDL > pYDEP > pYMEM > pYEEI. This altered binding behavior of mutant Btk SH2 domain likely leads to XLA.     

Quantitative relation between intermolecular and intramolecular binding of pro-rich peptides to SH3 domains

Flexible linkers are often found to tether binding sequence motifs or connect protein domains. Here we analyze three usages of flexible linkers: 1), intramolecular binding of proline-rich peptides (PRPs) to SH3 domains for kinase regulation; 2), intramolecular binding of PRP for increasing the folding stability of SH3 domains; and 3), covalent linking of PRPs and other ligands for high-affinity bivalent binding. The basis of these analyses is a quantitative relation between intermolecular and intramolecular binding constants. This relation has the form K(i) = K(e0)p for intramolecular binding and K(e) = K(e01)K(e02)p for bivalent binding. The effective concentration p depends on the length of the linker and the distance between the linker attachment points in the bound state. Several applications illustrate the usefulness of the quantitative relation. These include intramolecular binding to the Itk SH3 domain by an internal PRP and to a circular permutant of the alpha-spectrin SH3 domain by a designed PRP, and bivalent binding to the two SH3 domains of Grb2 by two linked PRPs. These and other examples suggest that flexible linkers and sequence motifs tethered to them, like folded protein domains, are also subject to tight control during evolution.     

Experiment and theory highlight role of native state topology in SH3 folding.

We use a combination of experiments, computer simulations and simple model calculations to characterize, first, the folding transition state ensemble of the src SH3 domain, and second, the features of the protein that determine its folding mechanism. Kinetic analysis of mutations at 52 of the 57 residues in the src SH3 domain revealed that the transition state ensemble is even more polarized than suspected earlier: no single alanine substitution in the N-terminal 15 residues or the C-terminal 9 residues has more than a two-fold effect on the folding rate, while such substitutions at 15 sites in the central three-stranded beta-sheet cause significant decreases in the folding rate. Molecular dynamics (MD) unfolding simulations and ab initio folding simulations on the src SH3 domain exhibit a hierarchy of folding similar to that observed in the experiments. The similarity in folding mechanism of different SH3 domains and the similar hierarchy of structure formation observed in the experiments and the simulations can be largely accounted for by a simple native state topology-based model of protein folding energy landscapes.     

Kinetics of structural changes in the relay loop and SH3 domain of myosin

The intrinsic fluorescence of smooth muscle myosin signals conformational changes associated with different catalytic states of the ATPase cycle. To elucidate this relationship, we have examined the pre-steady-state kinetics of nucleotide binding, hydrolysis, and product release in motor domain-essential light chain mutants containing a single endogenous tryptophan, either residue 512 in the rigid relay loop or residue 29 adjacent to the SH3 domain. The intrinsic fluorescence of W512 is sensitive to both nucleotide binding and hydrolysis, and appears to report structural changes at the active site, presumably through a direct connection with switch II. The intrinsic fluorescence of W29 is sensitive to nucleotide binding but not hydrolysis, and does not appear to be tightly linked with structural changes occurring at the active site. We propose that the SH3 domain may be sensitive to conformational changes in the lever arm through contacts with the essential light chain.     

Hydration and packing along the folding pathway of SH3 domains by pressure-dependent NMR

The volumetric properties associated with protein folding transitions reflect changes in protein packing and hydration of the states that participate in the folding reaction. Here, NMR spin relaxation techniques are employed to probe the folding-unfolding kinetics of two SH3 domains as a function of pressure so that the changes in partial molar volumes along the folding pathway can be measured. The two domains fold with rates that differ by approximately 3 orders of magnitude, so their folding dynamics must be probed using different NMR relaxation experiments. In the case of the drkN SH3 domain that folds via a two-state mechanism on a time scale of seconds, nitrogen magnetization exchange spectroscopy is employed, while for the G48M mutant of the Fyn SH3 domain where the folding occurs on the millisecond time scale (three-step reaction), relaxation dispersion experiments are utilized. The NMR methodology is extremely sensitive to even small changes in equilibrium and rate constants, so reliable estimates of partial molar volumes can be obtained using low pressures (1-120 bar), thus minimizing perturbations to any of the states along the folding reaction coordinate. The volumetric data that were obtained are consistent with a similar folding mechanism for both SH3 domains, involving early chain compaction to states that are at least partially hydrated. This work emphasizes the role of NMR spin relaxation in studying dynamic processes over a wide range of time scales.     

Stability Changes upon Mutation of Solvent- accessible Residues in Proteins Evaluated by Database-derived Potentials

The stability changes in peptides and proteins caused by the substitution of a single amino acid, which can be measured experimentally by the change in folding free energy, are evaluated here using effective potentials derived from known protein structures. The analysis is focused on mutations of residues that are accessible to the solvent. These represent in total 106 mutations, introduced at different sites in barnase, bacteriophage T4 lysozyme and chymotrypsin inhibitor 2, and in a synthetic helical peptide. Assuming that the mutations do not modify the backbone structure, the changes in folding free energies are computed using various types of database-derived potentials and are compared with the measured ones. Distance-dependent residue – residue potentials are found to be inadequate for estimating the stability changes caused by these mutations, as they are dominated by hydrophobic interactions, which do not play an essential role at the protein surface. On the contrary, the potentials based on backbone torsion angle propensities yield quite good results. Indeed, for a subset of 96 out of the 106 mutations, the computed and measured changes in folding free energy correlate with a linear correlation coefficient of 0.87. Moreover, the ten mutations that are excluded from the correlation either seem to cause modifications of the backbone structure or to involve strong hydrophobic interactions, which are atypical for solvent-accessible residues. We find furthermore that raising the ionic strength of the solvent used for measuring the changes in folding free energies improves the correlation, as it tends to mask the electrostatic interactions. When adding to these 106 mutations 44 mutations performed in staphylococcal nuclease and chemotactic protein, which were first discarded because some of them were suspected to affect the backbone conformation or the denatured state, the correlation between measured and computed folding free energy changes remains quite good: the correlation coefficient is 0.86 for 135 out of the 150 mutations. The success of the backbone torsion potentials in predicting stability changes indicates that the approximations made for deriving these potentials are adequate. It suggests moreover that the local interactions along the chain dominate at the protein surface.     

Folding of the alphaII-spectrin SH3 domain under physiological salt conditions

The SH3 domain has often been used as a model for protein folding due to its typical two-state behaviour. However, recent experimental data at low pH as well as molecular dynamic simulations have indicated that the folding process of SH3 probably is more complicated, and may involve intermediate states. Using both kinetic and equilibrium measurements we have obtained evidence that under native-like conditions the folding of the spectrin SH3 domain does not follow a classic two-state behaviour. The curvature we observed in the Chevron plots is a strong indication of a non-linear activation energy relationship due to the presence of high-energy intermediates. In addition, circular dichroism measurements indicated that refolding after thermal denaturation did not follow the same pattern as thermal unfolding but rather implied less cooperativity and that the refolding transition increased with increasing protein concentration. Further, NMR experiments indicated that upon refolding the SH3 domain gave rise to more than one conformation. Therefore, our results suggest that the folding of the SH3 domain of αII-spectrin does not follow a classical two-state process under high-salt conditions and neutral pH. Heterogeneous folding pathways, which can include folding intermediates as well as misfolded intermediates, might give a more reasonable insight into the folding behaviour of the αII-spectrin SH3 domain.     

Structure, dynamics, and Hck interaction of full-length HIV-1 Nef

Nef is an HIV accessory protein that plays an important role in the progression of disease after viral infection. It interferes with numerous signaling pathways, one of which involves serine/threonine kinases. Here, we report the results of an NMR structural investigation on full-length Nef and its interaction with the entire regulatory domain of Hck (residues 72-256; Hck32L). A helical conformation was found at the N-terminus for residues 14-22, preceding the folded core domain. In contrast to the previously studied truncated Nef (Nef Δ1-39), the full-length Nef did not show any interactions of Trp57/Leu58 with the hydrophobic patch formed by helices α1 and α2. Upon Hck32L binding, the N-terminal anchor domain as well as the well-known SH3-binding site of Nef exhibited significant chemical shift changes. Upon Nef binding, resonance changes in the Hck spectrum were confined mostly to the SH3 domain, with additional effects seen for the connector between SH3 and SH2, the N-terminal region of SH2 and the linker region that contains the regulatory polyproline motif. The binding data suggest that in full-length Nef more than the core domain partakes in the interaction. The solution conformation of Hck32L was modeled using RDC data and compared with the crystal structure of the equivalent region in the inactivated, full-length Hck, revealing a notable difference in the relative orientations of the SH3 and SH2 domains. The RDC-based model combined with (15)N backbone dynamics data suggest that Hck32L adopts an open conformation without binding of the polyproline motif in the linker to the SH3 domain.