Identification of a collapsed intermediate with non-native long-range interactions on the folding pathway of a pair of Fyn SH3 domain mutants by NMR relaxation dispersion spectroscopy

Recent 15N and 13C spin-relaxation dispersion studies of fast-folding mutants of the Fyn SH3 domain have established that folding proceeds through a low-populated on-pathway intermediate (I) where the central beta-sheet is at least partially formed, but without interactions between the NH2- and COOH-terminal beta-strands that exist in the folded state (F). Initial studies focused on mutants where Gly48 is replaced; in an effort to establish whether this intermediate is a general feature of Fyn SH3 folding a series of 15N relaxation experiments monitoring the folding of Fyn SH3 mutants N53P/V55L and A39V/N53P/V55L are reported here. For these mutants as well, folding proceeds through an on-pathway intermediate with similar features to those observed for G48M and G48V Fyn SH3 domains. However, the 15N chemical shifts extracted for the intermediate indicate pronounced non-native contacts between the NH2 and COOH-terminal regions not observed previously. The kinetic parameters extracted for the folding of A39V/N53P/V55L Fyn SH3 from the three-state folding model F<-->I<-->U are in good agreement with folding and unfolding rates extrapolated to zero denaturant obtained from stopped-flow experiments analyzed in terms of a simplified two-state folding reaction. The folding of the triple mutant was studied over a wide range of temperatures, establishing that there is no difference in heat capacities between F and I states. This confirms a compact folding intermediate structure, which is supported by the 15N chemical shifts of the I state extracted from the dispersion data. The temperature-dependent relaxation data simplifies data analysis because at low temperatures (< 25 degrees C) the unfolded state (U) is negligibly populated relative to I and F. A comparison between parameters extracted at low temperatures where the F<-->I exchange model is appropriate with those from the more complex, three-state model at higher temperatures has been used to validate the protocol for analysis of three-site exchange relaxation data.     

Side-chain interactions in the folding pathway of a Fyn SH3 domain mutant studied by relaxation dispersion NMR spectroscopy

A major challenge to the study of protein folding is the fact that intermediate states along the reaction pathway are generally unstable and thus difficult to observe. Recently developed NMR relaxation dispersion experiments present an avenue to accessing such states, providing kinetic, thermodynamic, and structural information for intermediates with small (greater than or equal to approximately 1%) populations at equilibrium. We have employed these techniques to study the three-state folding reaction of the G48M Fyn SH3 domain. Using (13)C-, (1)H-, and (15)N-based methods, we have characterized backbone and side-chain interactions in the folded, unfolded, intermediate, and transition states, thereby mapping the energy landscape of the protein. We find that the intermediate, populated to approximately 1%, contains nativelike structure in a central beta-sheet, and is disordered at the amino and carboxy termini. The intermediate is stabilized by side-chain van der Waals contacts, yet (13)C chemical shifts indicate that methyl-containing residues remain disordered. This state has a partially structured backbone and a collapsed yet mobile hydrophobic core and thus closely resembles a molten globule. Nonpolar side-chain contacts are formed in the unfolded-intermediate transition state; these interactions are disrupted in the intermediate-folded transition state, possibly allowing side chains to rearrange as they adopt the native packing configuration. This work illustrates the power of novel relaxation dispersion experiments in characterizing excited states that are "invisible" in even the most sensitive of NMR experiments.     

A 2D 13C-CEST experiment for studying slowly exchanging protein systems using methyl probes: an application to protein folding

A 2D (13)C Chemical Exchange Saturation Transfer (CEST) experiment is presented for studying slowly exchanging protein systems using methyl groups as probes. The utility of the method is first established through studies of protein L, a small protein, for which chemical exchange on the millisecond time-scale is not observed. Subsequently the approach is applied to a folding exchange reaction of a G48M mutant Fyn SH3 domain, for which only cross-peaks derived from the folded ('ground') state are present in spectra. Fits of (15)N and methyl (13)C CEST profiles of the Fyn SH3 domain establish that the exchange reaction involves an interchange between folded and unfolded conformers, although elevated methyl (13)C transverse relaxation rates for some of the residues of the unfolded ('invisible, excited') state indicate that it likely exchanges with a third conformation as well. In addition to the kinetics of the exchange reaction, methyl carbon chemical shifts of the excited state are also obtained from analysis of the (13)C CEST data.     

1H, 13C and 15N backbone and side-chain chemical shift assignment of the Fyn SH2 domain and its complex with a phosphotyrosine peptide

SH2 domains are interaction modules uniquely dedicated to recognize phosphotyrosine sites, playing a central role in for instance the activation of tyrosine kinases or phosphatases. Here we report the ¹H, ¹⁵N and ¹³C backbone and side-chain chemical shift assignments of the SH2 domain of the human protein tyrosine kinase Fyn, both in its free state and bound to a high-affinity phosphotyrosine peptide corresponding to a specific sequence in the hamster middle-T antigen. The BMRB accession numbers are 17,368 and 17,369, respectively.     

The folding pathway of an FF domain: characterization of an on-pathway intermediate state under folding conditions by (15)N, (13)C(alpha) and (13)C-methyl relaxation dispersion and (1)H/(2)H-exchange NMR spectroscopy

The FF domain from the human protein HYPA/FBP11 folds via a low-energy on-pathway intermediate (I). Elucidation of the structure of such folding intermediates and denatured states under conditions that favour folding are difficult tasks. Here, we investigated the millisecond time-scale equilibrium folding transition of the 71-residue four-helix bundle wild-type protein by (15)N, (13)C(alpha) and methyl(13)C Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion experiments and by (1)H/(2)H-exchange measurements. The relaxation data for the wild-type protein fitted a simple two-site exchange process between the folded state (F) and I. Destabilization of F in mutants A17G and Q19G allowed the detection of the unfolded state U by (15)N CPMG relaxation dispersion. The dispersion data for these mutants fitted a three-site exchange scheme, U<-->I<-->F, with I populated higher than U. The kinetics and thermodynamics of the folding reaction were obtained via temperature and urea-dependent relaxation dispersion experiments, along with structural information on I from backbone (15)N, (13)C(alpha) and side-chain methyl (13)C chemical shifts, with further information from protection factors for the backbone amide groups from (1)H/(2)H-exchange. Notably, helices H1-H3 are at least partially formed in I, while helix H4 is largely disordered. Chemical shift differences for the methyl (13)C nuclei suggest a paucity of stable, native-like hydrophobic interactions in I. These data are consistent with Phi-analysis of the rate-limiting transition state between I and F. The combination of relaxation dispersion and Phi data can elucidate whole experimental folding pathways.     

Assessment of the Effects of Increased Relaxation Dispersion Data on the Extraction of 3-site Exchange Parameters Characterizing the Unfolding of an SH3 Domain

Recently a suite of six CPMG relaxation dispersion experiments has been described for quantifying millisecond time-scale exchange processes in proteins. The methodology has been applied to study the folding reaction of a G48M Fyn SH3 domain mutant that exchanges between the native state, and low populated unfolded and intermediate states. A complex non-linear global optimization protocol allows extraction of the kinetics and thermodynamics of the 3-site exchange process from the experimental data, as well as reconstruction of the amide group chemical shifts of the excited states. We show here, through a series of Monte-Carlo simulations on various synthetic data sets, that the 3-site exchange parameters extracted for this system on the basis of ¹⁵N single-quantum (SQ) dispersion profiles exclusively, recorded at a single temperature, are significantly in error. While a temperature dependent ¹⁵N study improves the robustness of extracted parameters, as does a combined analysis of ¹⁵N and ¹H SQ data sets measured at a single temperature, the best agreement is observed in cases where the full complement of six dispersion profiles per residue is analyzed.     

Abp1p and Fyn SH3 domains fold through similar low-populated intermediate states

Src homology 3 (SH3) domains are small modules that are thought to fold via a two-state mechanism, without the accumulation of significant populations of intermediate states. Relaxation dispersion NMR studies of the folding of G48V and G48M mutants of the Fyn SH3 domain have established that, at least for these modules, folding proceeds through the formation of a transient on-pathway intermediate with an equilibrium population of 1-2% that can be readily detected [Korzhnev, D. M., et al. (2004) Nature 430, 586-590]. To investigate the generality of this result, we present an (15)N relaxation dispersion NMR study of a pair of additional SH3 domains, including a G48V mutant of a stabilized Abp1p SH3 domain that shares 36% sequence identity with the Fyn SH3 module, and a A39V/N53P/V55L mutant Fyn SH3 domain. A transient folding intermediate is detected for both of the proteins studied here, and the dispersion data are well fit to a folding model of the form F <--> I <--> U, where F, I, and U correspond to folded, intermediate, and unfolded states, respectively. The temperature dependencies of the folding/unfolding rate constants were obtained so that the thermodynamic properties of each of F, I, and U could be established. The detection of I states in folding pathways of all SH3 domains examined to date via relaxation dispersion NMR spectroscopy indicates that such intermediates may well be a conserved feature in the folding of such domains in general but that their transient nature along with their low population makes detection difficult using more well-established approaches to the study of folding.     

Cross-correlated spin relaxation effects in methyl <Superscript>1</Superscript>H CPMG-based relaxation dispersion experiments: Complications and a simple solution

Artifacts associated with the measurement of methyl ¹H single quantum CPMG-based relaxation dispersion profiles are described. These artifacts arise due to the combination of cross-correlated spin relaxation effects involving intra-methyl ¹H–¹H dipolar interactions and imperfections in ¹H refocusing pulses that are applied during CPMG intervals that quantify the effects of chemical exchange on measured transverse relaxation rates. As a result substantial errors in extracted exchange parameters can be obtained. A simple work-around is presented where the ¹H chemical shift difference between the exchanging states is extracted from a combination of ¹³C single quantum and ¹³C–¹H multiple quantum dispersion profiles. The approach is demonstrated with an application to a folding/unfolding reaction involving a G48M mutant Fyn SH3 domain.     

Assignment of backbone 1H, 13C,and 15N resonances of the SH2 domain of human Grb14

The ¹H, ¹³C, and ¹⁵N backbone resonance assignments have been made for the Src homology 2 (SH2) domain of the human molecular adapter protein Grb14. The assignments, along with the majority of the non-aromatic side-chain ¹H and ¹³C resonances are reported. The SH2 domain has been complexed with a phosphotyrosine-containing peptide (pY766) corresponding to the putative binding site in the fibroblast growth factor receptor (FGFR1). Chemical shift changes upon binding are also reported.     

F NMR studies of solvent exposure and peptide binding to an SH3 domain

¹⁹F NMR was used to study topological features of the SH3 domain of Fyn tyrosine kinase for both the free protein and a complex formed with a binding peptide. Metafluorinated tyrosine was biosynthetically incorporated into each of 5 residues of the G48M mutant of the SH3 domain (i.e. residues 8, 10, 49 and 54 in addition to a single residue in the linker region to the C-terminal polyhistidine tag). Distinct ¹⁹F NMR resonances were observed and subsequently assigned after separately introducing single phenylalanine mutations. ¹⁹F NMR chemical shifts were dependent on protein concentration above 0.6 mM, suggestive of dimerization via the binding site in the vicinity of the tyrosine side chains. ¹⁹F NMR spectra of Fyn SH3 were also obtained as a function of concentration of a small peptide (2-hydroxynicotinic-NH)–Arg–Ala–Leu–Pro–Pro–Leu–Pro-diaminopropionic acid –NH₂, known to interact with the canonical polyproline II (PPII) helix binding site of the SH3 domain. Based on the ¹⁹F chemical shifts of Tyr8, Tyr49, and Tyr54, as a function of peptide concentration, an equilibrium dissociation constant of 18 ± 4 μM was obtained. Analysis of the line widths suggested an average exchange rate, k_ex, associated with the peptide–protein two-site exchange, of 5200 ± 600 s^− 1 at a peptide concentration where 96% of the FynSH3 protein was assumed to be bound. The extent of solvent exposure of the fluorine labels was studied by a combination of solvent isotope shifts and paramagnetic effects from dissolved oxygen. Tyr54, Tyr49, Tyr10, and Tyr8, in addition to the Tyr on the C-terminal tag, appear to be fully exposed to the solvent at the metafluoro position in the absence of binding peptide. Tyr54 and, to some extent, Tyr10 become protected from the solvent in the peptide bound state, consistent with known structural data on SH3–domain peptide complexes. These results show the potential utility of ¹⁹F-metafluorotyrosine to probe protein–protein interactions in conjunction with paramagnetic contrast agents.     

Population shuffling between ground and high energy excited states

Stochastic processes powered by thermal energy lead to protein motions traversing time-scales from picoseconds to seconds. Fundamental to protein functionality is the utilization of these dynamics for tasks such as catalysis, folding, and allostery. A hierarchy of motion is hypothesized to connect and synergize fast and slow dynamics toward performing these essential activities. Population shuffling predicts a “top-down” temporal hierarchy, where slow time-scale conformational interconversion leads to a shuffling of the free energy landscape for fast time-scale events. Until now, population shuffling was only applied to interconverting ground states. Here, we extend the framework of population shuffling to be applicable for a system interconverting between low energy ground and high energy excited states, such as the SH3 domain mutants G48M and A39V/N53P/V55L from the Fyn tyrosine kinase, providing another tool for accessing the structural dynamics of high energy excited states. Our results indicate that the higher energy gauche− rotameric state for the leucine χ₂ dihedral angle contributes significantly to the distribution of rotameric states in both the major and minor forms of the SH3 domain. These findings are corroborated with unrestrained molecular dynamics (MD) simulations on both the major and minor states of the SH3 domain demonstrating high correlations between experimental and back-calculated leucine χ₂ rotameric populations. Taken together, we demonstrate how fast time-scale rotameric side-chain population distributions can be extracted from slow time-scale conformational exchange data further extending the scope and the applicability of the population shuffling model.     

1H, 13C and 15N NMR assignments and secondary structure of the paramagnetic form of rat cytochrome b 5

Summary Modern multidimensional double- and triple-resonance NMR methods have been applied to assign the backbone and side-chain ¹³C resonances for both equilibrium conformers of the paramagnetic form of rat liver microsomal cytochrome b ₅. The assignment of backbone ¹³C resonances was used to confirm previous ¹H and ¹⁵N resonance assignments [Guiles, R.D. et al. (1993) Biochemistry, 32, 8329–8340]. On the basis of short- and medium-range NOEs and backbone ¹³C chemical shifts, the solution secondary structure of rat cytochrome b ₅ has been determined. The striking similarity of backbone ¹³C resonances for both equilibrium forms strongly suggests that the secondary structures of the two isomers are virtually identical. It has been found that the ¹³C chemical shifts of both backbone and side-chain atoms are relatively insensitive to paramagnetic effects. The reliability of such methods in anisotropic paramagnetic systems, where large pseudocontact shifts can be observed, is evaluated through calculations of the magnitude of such shifts.Abbreviations DANTE delays alternating with nutation for tailored excitation - DEAE diethylaminoethyl - DQF-COSY 2D double-quantum-filtered correlation spectroscopy - EDTA ethylenediaminetetraacetic acid - HCCH-TOCSY 3D proton-correlated carbon TOCSY experiment - HMQC 2D heteronuclear multiple-quantum correlation spectroscopy - HNCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons - HNCO 3D triple-resonance experiment correlating amide protons, amide nitrogens and carbonyl carbons - HNCOCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons via carbonyl carbons - HOHAHA 2D homonuclear Hartmann-Hahn spectroscopy - HOHAHA-HMQC 3D HOHAHA relayed HMQC - HSQC 2D heteronuclear single-quantum correlation spectroscopy - IPTG isopropyl thiogalactoside - NOESY 2D nuclear Overhauser enhancement spectroscopy - NOESY-HSQC 3D NOESY relayed HSQC - TOCSY 2D total correlation spectroscopy - TPPI time-proportional phase incrementation - TSP trimethyl silyl propionate     

Propensity to Form Amyloid Fibrils Is Encoded as Excitations in the Free Energy Landscape of Monomeric Proteins

Protein aggregation, linked to many of diseases, is initiated when monomers access rogue conformations that are poised to form amyloid fibrils. We show, using simulations of src SH3 domain, that mechanical force enhances the population of the aggregation-prone (N^?) states, which are rarely populated under force free native conditions but are encoded in the spectrum of native fluctuations. The folding phase diagrams of SH3 as a function of denaturant concentration ([C]), mechanical force (f), and temperature exhibit an apparent two-state behavior, without revealing the presence of the elusive N^? states. Interestingly, the phase boundaries separating the folded and unfolded states at all [C] and f fall on a master curve, which can be quantitatively described using an analogy to superconductors in a magnetic field. The free energy profiles as a function of the molecular extension (R), which are accessible in pulling experiments, (R), reveal the presence of a native-like N^? with a disordered solvent-exposed amino-terminal β-strand. The structure of the N^? state is identical with that found in Fyn SH3 by NMR dispersion experiments. We show that the timescale for fibril formation can be estimated from the population of the N^? state, determined by the free energy gap separating the native structure and the N^? state, a finding that can be used to assess fibril forming tendencies of proteins. The structures of the N^? state are used to show that oligomer formation and likely route to fibrils occur by a domain-swap mechanism in SH3 domain.     

The high resolution NMR structure of the third SH3 domain of CD2AP

CD2 associated protein (CD2AP) is an adaptor protein that plays an important role in cell to cell union needed for the kidney function. CD2AP interacts, as an adaptor protein, with different natural targets, such as CD2, nefrin, c-Cbl and podocin. These proteins are believed to interact to one of the three SH3 domains that are positioned in the N-terminal region of CD2AP. To understand the network of interactions between the natural targets and the three SH3 domains (SH3-A, B and C), we have started to determine the structures of the individual SH3 domains. Here we present the high-resolution structure of the SH3-C domain derived from NMR data. Full backbone and side-chain assignments were obtained from triple-resonance spectra. The structure was determined from distance restraints derived from high-resolution 600 and 800 MHz NOESY spectra, together with phi and psi torsion angle restraints based on the analysis of ¹HN, ¹⁵N, ¹Hα, ¹³Cα, ¹³CO and ¹³Cβ chemical shifts. Structures were calculated using CYANA and refined in water using RECOORD. The three-dimensional structure of CD2AP SH3-C contains all the features that are typically found in other SH3 domains, including the general binding site for the recognition of polyproline sequences.     

Structure and Disorder in an Unfolded State under Nondenaturing Conditions from Ensemble Models Consistent with a Large Number of Experimental Restraints

Obtaining detailed structural models of disordered states of proteins under nondenaturing conditions is important for a better understanding of both functional intrinsically disordered proteins and unfolded states of folded proteins. Extensive experimental characterization of the drk N-terminal SH3 domain unfolded state has shown that, although it appears to be highly disordered, it possesses significant nonrandom secondary and tertiary structure. In our previous attempts to generate structural models of the unfolded state using the program ENSEMBLE, we were limited by insufficient experimental restraints and conformational sampling. In this study, we have vastly expanded our experimental restraint set to include ¹H-¹⁵N residual dipolar couplings, small-angle X-ray scattering measurements, nitroxide paramagnetic relaxation enhancements, O₂-induced ¹³C paramagnetic shifts, hydrogen-exchange protection factors, and ¹⁵N R₂ data, in addition to the previously used nuclear Overhauser effects, amino terminal Cu²⁺-Ni²⁺ binding paramagnetic relaxation enhancements, J-couplings, chemical shifts, hydrodynamic radius, and solvent accessibility restraints. We have also implemented a new ensemble calculation methodology that uses iterative conformational sampling and seeks to calculate the simplest possible ensemble models. As a result, we can now generate ensembles that are consistent with much larger experimental data sets than was previously possible. Although highly heterogeneous and having broad molecular size distributions, the calculated drk N-terminal SH3 domain unfolded-state ensembles have very different properties than expected for random or statistical coils and possess significant nonnative α-helical structure and both native-like and nonnative tertiary structure.     

The SH2 domain is crucial for function of Fyn in neuronal migration and cortical lamination

Neurons in the developing brain form the cortical plate (CP) in an inside-out manner, in which the late-born neurons are located more superficially than the early-born neurons. Fyn, a member of the Src family kinases, plays an important role in neuronal migration by binding to many substrates. However, the role of the Src-homology 2 (SH2) domain in function of Fyn in neuronal migration remains poorly understood. Here, we demonstrate that the SH2 domain is essential for the action of Fyn in neuronal migration and cortical lamination. A point mutation in the Fyn SH2 domain (Fyn^R176A) impaired neuronal migration and their final location in the cerebral cortex, by inducing neuronal aggregation and branching. Thus, we provide the first evidence of the Fyn SH2 domain contributing to neuronal migration and neuronal morphogenesis. [BMB Reports 2015; 48(2): 97-102]     

HCV NS5A Protein Containing Potential Ligands for Both Src Homology 2 and 3 Domains Enhances Autophosphorylation of Src Family Kinase Fyn in B Cells

Hepatitis C virus (HCV) infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin''s lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A) protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV) implied that NS5A was tyrosine phosphorylated by pervanadate (PV) treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST)-fusion proteins of various Src homology 2 (SH2) domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3) domain. Substitution of Arg¹⁷⁶ to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr³³⁴ was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.