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Biomolecular condensates concentrate proteins, nucleic acids, and small molecules and play an essential role in many biological processes. Their formation is tuned by a balance between energetically favorable and unfavorable contacts, with charge-charge interactions playing a central role in some systems. The positively charged intrinsically disordered carboxy-terminal region of the RNA-binding protein CAPRIN1 is one such example, phase separating upon addition of negatively charged ATP or high concentrations of sodium chloride (NaCl). Using solution NMR spectroscopy, we measured residue-specific near-surface electrostatic potentials (ϕ

Stress granules and P-bodies are cytosolic biomolecular condensates that dynamically form by the phase separation of RNAs and proteins. They participate in translational control and buffer the proteome. Upon stress, global translation halts and mRNAs bound to the translational machinery and other proteins coalesce to form stress granules (SGs). Similarly, translationally stalled mRNAs devoid of translation initiation factors shuttle to P-bodies (PBs). Here, we review the cumulative progress made in defining the protein components that associate with mammalian SGs and PBs. We discuss the composition of SG and PB proteomes, supported by a new user-friendly database (http://rnagranuledb.lunenfeld.ca/) that curates current literature evidence for genes or proteins associated with SGs or PBs. As previously observed, the SG and PB proteomes are biased toward intrinsically disordered regions and have a high propensity to contain primary sequence features favoring phase separation. We also provide an outlook on how the various components of SGs and PBs may cooperate to organize and form membraneless organelles.

Conformational states of the metastable drkN SH3 domain were characterized using single-molecule fluorescence techniques. Under nondenaturing conditions, two Förster resonance energy transfer (FRET) populations were observed that corresponded to a folded and an unfolded state. FRET-estimated radii of gyration and hydrodynamic radii estimated by fluorescence correlation spectroscopy of the two coexisting conformations are in agreement with previous ensemble x-ray scattering and NMR measurements. Surprisingly, when exposed to high concentrations of urea and GdmCl denaturants, the protein still exhibits two distinct FRET populations. The dominant conformation is expanded, showing a low FRET efficiency, consistent with the expected behavior of a random chain with excluded volume. However, approximately one-third of the drkN SH3 conformations showed high, nearly 100%, FRET efficiency, which is shown to correspond to denaturation-induced looped conformations that remain stable on a timescale of at least 100 μs. These loops may contain interconverting conformations that are more globally collapsed, hairpin-like, or circular, giving rise to the observed heterogeneous broadening of this population. Although the underlying mechanism of chain looping remains elusive, FRET experiments in formamide and dimethyl sulfoxide suggest that interactions between hydrophobic groups in the distal regions may play a significant role in the formation of the looped state.

Microexons represent the most highly conserved class of alternative splicing, yet their functions are poorly understood. Here, we focus on closely related neuronal microexons overlapping prion-like domains in the translation initiation factors, eIF4G1 and eIF4G3, the splicing of which is activity dependent and frequently disrupted in autism. CRISPR-Cas9 deletion of these microexons selectively upregulates synaptic proteins that control neuronal activity and plasticity and further triggers a gene expression program mirroring that of activated neurons. Mice lacking the Eif4g1 microexon display social behavior, learning, and memory deficits, accompanied by altered hippocampal synaptic plasticity. We provide evidence that the eIF4G microexons function as a translational brake by causing ribosome stalling, through their propensity to promote the coalescence of cytoplasmic granule components associated with translation repression, including the fragile X mental retardation protein FMRP. The results thus reveal an autism-disrupted mechanism by which alternative splicing specializes neuronal translation to control higher order cognitive functioning.

Liquid granules rich in intrinsically disordered proteins and RNA play key roles in critical cellular functions such as RNA processing and translation. Many details of the mechanism via which this occurs still remain to be elucidated. Motivated by the lacuna in the field, and by the prospects of developing de novo artificial granules that provide extrinsic control of translation, we report a bottom-up approach to engineer ribonucleoprotein granules comprised of a recombinant RNA-binding IDP that exhibits phase behavior in water. We developed a kinetic model to illustrate that these granules inhibit translation through reversible or irreversible sequestration of mRNA. Within monodisperse droplets capable of transcription and translation, we experimentally demonstrate temporal inhibition of translation, by using designer IDPs that exhibit tunable phase behavior. This work lays the foundation for developing artificial granules that promise to further our mechanistic understanding of their naturally occurring counterparts.

SummaryCap-dependent translation initiation is regulated by the interaction of eukaryotic initiation factor 4E (eIF4E) with eIF4E binding proteins (4E-BPs). Whereas the binding of 4E-BP peptides containing the eIF4E-binding 54YXXXXLΦ60 motif has been studied, atomic-level characterization of the interaction of eIF4E with full-length 4E-BPs has been lacking. Here, we use isothermal titration calorimetry and nuclear magnetic resonance spectroscopy to characterize the dynamic, structural and binding properties of 4E-BP2. Although disordered, 4E-BP2 contains significant fluctuating secondary structure and binds eIF4E at an extensive bipartite interface including the canonical 54YXXXXLΦ60 and 78IPGVT82 sites. Each of the two binding elements individually has submicromolar affinity and exchange on and off of the eIF4E surface within the context of the overall nanomolar complex. This dynamic interaction facilitates exposure of regulatory phosphorylation sites within the complex. The 4E-BP2 interface on eIF4E overlaps yet is more extensive than the eIF4G:eIF4E interface, suggesting that these key interactions may be differentially targeted for therapeutics.

Sic1 is a cyclin-dependent kinase inhibitor which must be phosphorylated on at least six sites (termed Cdc4 phosphodegrons, CPDs) to allow its recognition by the WD40 binding domain of Cdc4. The highly-cooperative switch-like dependence on the number of phosphorylated sites on Sic1 cannot be accounted for by traditional thermodynamic models of cooperativity. Further experimental attention is necessary to determine the physicochemical/mechanistic basis of its highly cooperative binding.We used single molecule fluorescence techniques to study the dimensions and dynamics of Sic1's N-terminal targeting region (residues 1-90, henceforth Sic1), phosphorylated Sic1 (pSic1), and the pSic1-WD40 dynamic complex. Using time-resolved fluorescence anisotropy, we find local segment specific rotational correlation times which are complexly modulated by chain compactness and electrostatics (charge screening and phosphorylation).Previous single molecule Fӧrster Resonance Energy Transfer (smFRET) measurements [1] observed end-to-end reconfiguration on timescales larger than ∼1ms; resulting in FRET histograms with multiple conformational sub-ensembles. These sub-ensembles and their dynamics are further explored by trapping single Sic1, pSic1 and WD40 in nanometer sized surface-tethered lipid vesicles and modulating their electrostatics and compactness. In a refinement to the conventional approaches for inferring dimensions from smFRET experiments, we use distance distributions from Monte Carlo simulations which extensively sample coarse-grained protein conformations. The application of polymer physics theory/simulation towards smFRET data interpretation, and towards IDP binding, contributes to the growing toolkit for understanding the diverse behaviours of IDPs.[1] Liu B. et al., J. Phys. Chem. B. 2014 118(15):4088-97.

Sic1 is a cyclin-dependent kinase inhibitor which must be phosphorylated on at least six sites (termed Cdc4 phosphodegrons, CPDs) to allow its recognition by the WD40 binding domain of Cdc4. The highly-cooperative switch-like dependence on the number of phosphorylated sites on Sic1 cannot be accounted for by traditional thermodynamic models of cooperativity. Further experimental attention is necessary to determine the physicochemical/mechanistic basis of its highly cooperative binding.We used single molecule fluorescence techniques to study the dimensions and dynamics of Sic1's N-terminal targeting region (residues 1-90, henceforth Sic1), phosphorylated Sic1 (pSic1), and the pSic1-WD40 dynamic complex.Previous single molecule Fӧrster Resonance Energy Transfer (smFRET) measurements [Liu, 2014] observed end-to-end reconfiguration on timescales larger than ∼1ms; resulting in FRET histograms with multiple conformational sub-ensembles. Sic1, pSic1, and the pSic1-WD40 complex are examined using smFRET to study the dynamics and dimensions of the various sub-ensembles. In a refinement to the conventional approaches for inferring dimensions from smFRET experiments, we use distance distributions from Monte Carlo simulations which extensively sample coarse-grained protein conformations. The application of polymer physics theory/simulation towards smFRET data interpretation, and towards IDP binding, contributes to the growing toolkit for understanding the diverse behaviours of IDPs.

Complete folding is not a prerequisite for protein function, as disordered and partially folded states of proteins frequently perform essential biological functions. In order to understand their functions at the molecular level, we utilized diverse experimental measurements to calculate ensemble models of three non-homologous, intrinsically disordered proteins: I-2, spinophilin and DARPP-32, which bind to and regulate protein phosphatase 1 (PP1). The models demonstrate that these proteins have dissimilar propensities for secondary and tertiary structure in their unbound forms. Direct comparison of these ensemble models with recently determined PP1 complex structures suggests a significant role for transient, pre-formed structure in the interactions of these proteins with PP1. Finally, we generated an ensemble model of partially disordered I-2 bound to PP1 that provides insight into the relationship between flexibility and biological function in this dynamic complex.

Summary Many regulatory proteins, including the transcription factor c-Jun, are highly enriched in disordered protein regions that govern growth, division, survival, differentiation, and response to signals. The stability of c-Jun is controlled by poorly understood regulatory interactions of its disordered region with both the E3 ubiquitin ligase SCF Fbw7 and prolyl cis - trans isomerase Pin1. We use nuclear magnetic resonance and fluorescence studies of c-Jun to demonstrate that multisite c-Jun phosphorylation is required for high-affinity interaction with Fbw7. We show that the Pin1 WW and PPIase domains interact in a dynamic complex with multiply phosphorylated c-Jun. Importantly, Pin1 isomerizes a pSer-Pro peptide bond at the c-Jun N terminus that affects binding to Fbw7 and thus modulates the ubiquitin-mediated degradation of c-Jun. Our findings support the general principle that multiple weak binding motifs within disordered regions can synergize to yield high-affinity interactions and provide rapidly evolvable means to build and fine-tune regulatory events.

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 (1)H-(15)N residual dipolar couplings, small-angle X-ray scattering measurements, nitroxide paramagnetic relaxation enhancements, O(2)-induced (13)C paramagnetic shifts, hydrogen-exchange protection factors, and (15)N R(2) data, in addition to the previously used nuclear Overhauser effects, amino terminal Cu(2+)-Ni(2+) 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 alpha-helical structure and both native-like and nonnative tertiary structure.

(19)F 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 (19)F NMR resonances were observed and subsequently assigned after separately introducing single phenylalanine mutations. (19)F 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. (19)F 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 -NH(2), known to interact with the canonical polyproline II (PPII) helix binding site of the SH3 domain. Based on the (19)F chemical shifts of Tyr8, Tyr49, and Tyr54, as a function of peptide concentration, an equilibrium dissociation constant of 18 +/- 4 microM 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 (19)F-metafluorotyrosine to probe protein-protein interactions in conjunction with paramagnetic contrast agents.

Smurf2 is an E3 ubiquitin ligase that drives degradation of the transforming growth factor-beta receptors and other targets. Recognition of the receptors by Smurf2 is accomplished through an intermediary protein, Smad7. Here we have demonstrated that the WW3 domain of Smurf2 can directly bind to the Smad7 polyproline-tyrosine (PY) motif. Of particular interest, the highly conserved WW domain binding site Trp, which interacts with target PY motifs, is a Phe in the Smurf2 WW3 domain. To examine this interaction, the solution structure of the complex between the Smad7 PY motif region (ELESPPPPYSRYPMD) and the Smurf2 WW3 domain was determined. The structure reveals that, in addition to binding the PY motif, the WW3 domain binds six residues C-terminal to the PY motif (PY-tail). Although the Phe in the WW3 domain binding site decreases affinity relative to the canonical Trp, this is balanced by additional interactions between the PY-tail and the beta1-strand and beta1-beta2 loop of the WW3 domain. The interaction between the Smurf2 WW3 domain and the Smad7 PY motif is the first example of PY motif recognition by a WW domain with a Phe substituted for the binding site Trp. This unusual interaction allows the Smurf2 WW3 domain to recognize a subset of PY motif-containing proteins utilizing an expanded surface to provide specificity.

SummaryInteractions between the WW domains of Drosophila Nedd4 (dNedd4) and Commissureless (Comm) PY motifs promote axon crossing at the CNS midline and muscle synaptogenesis. Here we report the solution structure of the dNedd4 WW3∗ domain complexed to the second PY motif (227′TGLPSYDEALH237′) of Comm. Unexpectedly, there are interactions between WW3∗ and ligand residues both N- and C-terminal to the PY motif. Residues Y232′–L236′ form a helical turn, following the PPII helical PY motif. Mutagenesis and binding studies confirm the importance of these extensive contacts, not simultaneously observed in other WW domain complexes, and identify a variable loop in WW3∗ responsible for its high-affinity interaction. These studies expand our general understanding of the molecular determinants involved in WW domain-ligand recognition. In addition, they provide insights into the specific regulation of dNedd4-mediated ubiquitination of Comm and subsequent internalization of Comm or the Comm/Roundabout complex, critical for CNS and muscle development.

In this issue of Structure, Conicella et al. (2016) present evidence that the low complexity C-terminal region of TDP-43 undergoes liquid-liquid phase separation. ALS-associated mutations alter this phase separation process, providing a possible mechanism for the pathology caused by these TDP-43 mutations. The work is strongly supportive of toxic loss of RNA processing function in ALS.

Intrinsically disordered proteins (IDPs) play critical roles in regulatory protein interactions. Cap-dependent translation initiation is regulated by the interaction of eukaryotic initiation factor 4E (eIF4E) with disordered eIF4E binding proteins (4E-BPs) in a phosphorylation dependent manner. Single molecule fluorescence resonance energy transfer (smFRET), fluorescence correlation spectroscopy (FCS), time-resolved fluorescence anisotropy (TRFA) and nuclear magnetic resonance (NMR) were used to detect and assess the structural changes and sequence-specific local chain motions of 4E-BP2 upon phosphorylation and upon binding to eIF4E.1H - 15N heteronuclear single quantum correlation (HSQC) spectra demonstrate that cysteine mutations of 4E-BP2 do not perturb its phosphorylated folded structure. Multiparameter smFRET analysis reveals changes in the conformational ensemble upon phosphorylation, denaturation and binding to elF4E. Nanosecond scale dynamics in 4E-BP2 were observed by site-specific FCS, and were tentatively assigned to formation of transient intrachain contacts. Our data suggests that multi-site phosphorylation of the protein slows down the proximal chain motions and also modulates the kinetics of distal regions. Segmental rotational correlation times and wobbling cone angles extracted for different sites along the chain provide a rigidity map of this IDP and can be used to evaluate its binding mode to eIF4E.

In yeast, the cyclin-dependent kinase inhibitor Sic1 is a disordered protein which interacts with a single site on its acceptor Cdc4 only upon multi-phosphorylation of its dispersed sites. To gain insight into the multi-phosphorylation dependence in Sic1-Cdc4 interaction, the conformational properties of the disordered Sic1 N-terminal targeting region were studied using single-molecule fluorescence spectroscopy.At least two Sic1 conformational populations with different sensitivities to charge screening by non-denaturing salts and ionic denaturants were identified. As described by the polyelectrolyte theory, the chain dimensions decrease monotonically with salt concentration and roll over at high denaturant, although a scaling factor of 1.2 indicates that Sic1 is not accurately described as a random chain. Fluorescence correlation analysis shows that Sic1 structure fluctuations occur on fast (10-100 ns) and slow (10-100 ms) ranges, with the fast phase being absent at low salt. Our data provides direct evidence that intrachain charge repulsions are significant for the conformational landscape of Sic1, and support the role of electrostatics in determining the size and shape of intrinsically-disordered proteins.View Large Image | View Hi-Res Image | Download PowerPoint Slide

For intrinsically disordered proteins (IDPs) containing a high density of charged residues, a Gaussian chain is an inadequate description of the protein's shape. One such IDP is Sic1, a highly positively charged IDP in the budding yeast Saccharomyces Cerevisiae which prevents the cell cycle from entering the S-phase from the G1-phase. Sic1's binding affinity for Cdc4 is highly phosphorylation state dependent, although the corresponding physical basis is not fully understood. NMR data supports the presence of a dynamic complex of Sic1:Cdc4 and a poly-electrostatic model has been proposed by Forman-Kay and coworkers.We studied the Sic1 N-terminal targeting region (1-90) to better understand the role of intrachain electrostatics, and to compare with the structural ensembles calculated from NMR and SAXS data. Sic1 is characterized using time-resolved fluorescence anisotropy (TRFA), which is sensitive to rotational and conformational dynamics. Sic1 exists in a dynamic ensemble of conformations and ensemble-averaged experiments have limitations in identifying and characterizing static and/or dynamic inhomogeneity in the motional dynamics. Therefore, we also performed burst spectroscopy and single-molecule TRFA on freely diffusing Sic1. Shape and flexibility were probed by varying the degree of intrachain repulsion screening through adjusting salt concentrations and described within a polymer physics framework, the polyelectrolyte model. To investigate the relative contributions of “global rotation” and “conformational flexibility”, Sic1 was labelled at three different sites. Sic1 is found to be well modelled as a prolate ellipsoid with internal flexibility.The single-molecule derived TRFA distribution data may be valuable as a constraint incorporated in future conformational ensemble calculations, complementary to SAXS and NMR data. Additionally, these measurements raise questions about the accuracy of highly charged IDP's radii of gyration when calculated from sm-FRET derived end-to-end distances, which often assume a Gaussian chain model for the end-to-end distance probability distribution.

Intrinsically disordered proteins and regions (IDPs/IDRs), which do not have stable secondary and tertiary structure, are capable of adopting different structural states. Many IDPs/IDRs populate conformationally heterogeneous monomeric states or engage in discrete interactions with other proteins, leading to folding upon binding or retaining significant disorder in the bound state. Others are involved in large-scale association having different degrees of order, from more defined fibers, to variably networked gels and to disordered liquid demixed states or droplets. These latter have been suggested to provide the physical basis for cellular membrane-less organelles such as the nucleolus.We have studied the N-terminal disordered region of Ddx4, an RNA DEAD-box helicase that is essential for formation of a class of membrane-less organelles termed nuage or germ granules functioning in spermatogenesis. When expressed in HeLa cells, the protein forms spherical, micron-sized, liquid-like cellular organelles. In vitro, it phase separates to form droplets with similar morphological and dynamic properties to the organelles observed in cells. Phase separation is sensitive to salt, pointing to the importance of electrostatic interactions. The sequence features of the disordered N-terminus of Ddx4 that underlie phase separation include clustering of charged residues into blocks of net positive and negative charge, with over-representation of FG/GF pairs and RG/GR pairs within the positive blocks. Perturbations of these properties disrupt phase separation, pointing to multi-valent cation-pi interactions playing an important role. The transient sampling of multi-valent interactions in self-association of Ddx4 extends previous observations of dynamic multi-valent interactions in discrete complexes of IDPs/IDRs, such as for Sic1:Cdc4 binding. The insights obtained from these and ongoing biophysical studies of Ddx4 will be valuable for developing a general understanding of the biogenesis and disassembly of membrane-less cellular organelles.

SH3 domains are highly involved in signal transduction and cellular localization. The N-terminal SH3 domain of Drosophila adaptor protein Drk is found to be marginally stable, exchanging between folded and unfolded states under non-denaturating conditions. The high unfolded state population makes DrkN SH3 a useful model system to study the physical polymeric properties of disordered protein states and to advance the understanding of the mechanism of protein folding.Single-molecule techniques have the unique capability to resolve populations of protein conformations and also the rates of exchange dynamics among them. Here, single-molecule Foster resonance energy transfer (smFRET) is performed in order to study the conformational distribution and dynamics of the DrkN SH3 domain, using fluorophores attached to two cysteines mutated at the N- and the C-termini of the 61amino acid chain. Freely-diffusing proteins in diluted solutions give rise to fluorescence bursts which can be quantitatively characterized. Multiparameter fluorescence analysis reveals two populations with different end-to-end distances, attributed to the folded and unfolded states coexisting under normal conditions. Conformational populations and internal chain dynamics are measured in both physiological and non-physiological conditions in order to understand the role of solvent-protein interactions for the structural stability. Fluorescence correlation spectroscopy (FCS) and FRET-FCS are applied to investigate local chain dynamics and the inter-conversion kinetics between the ordered and disordered conformations of DrkN SH3 in different solvent conditions.

Cystic fibrosis-causing mutations in CFTR can lead to protein misprocessing or channel gating defects, perturbing the ion and fluid flow across epithelial surfaces. CFTR transmembrane helices extend into the cytoplasm and interface with two nucleotide-binding domains, NBD1 and NBD2, via short 'coupling helices' (CLs). Channel gating is regulated by ATP binding and hydrolysis in the NBD1/NBD2 dimer and by phosphorylation of the NBD1 regulatory insert (RI) and R region immediately C-terminal of NBD1. We have used NMR spectroscopy to probe changes in conformational populations caused by ligand binding or mutation, revealing an allosteric network linking the CL4 binding site to regions near the RI and the R region. Conformational exchange occurs on multiple timescales in the NBD1 αRI construct, including μs-ms exchange and elevated flexibility in loops and C-terminal helices H8 and H9. Q637R, a mutation between H8 and H9 near to where the R region begins, changes dynamics in the RI deletion site and the CL4 binding site. VX-809, a drug that partially rescues both folding and gating defects, binds to the β-strands beneath H8/H9 of full-length NBD1, destabilizing these helices and perturbing some chemical shifts in the CL4 binding site. Titration of a CL4 peptide results in remote changes to H8/H9 and correlated chemical shifts observed at the RI. An allosteric network coupling the CL4 binding site to regions near both of the phospho-regulatory sites may contribute to the folding and gating defects that arise from mutations at the CL4:NBD1 interface, including those of the most common CF-causing mutation, F508del. Coupling of remote NBD1 sites by the observed allosteric network suggests that distinct inputs are integrated in CFTR processing and function and implies that correlation of NBD-ICL interactions might be achieved through targeting of these allosterically-linked sites.

The global fold of maltose-binding protein in complex with the substrate beta-cyclodextrin was determined by solution NMR methods. The two-domain protein is comprised of a single polypeptide chain of 370 residues, with a molecular mass of 42 kDa. Distance information in the form of H(N)-H(N), H(N)-CH(3) and CH(3)-CH(3) NOEs was recorded on (15)N, (2)H and (15)N, (13)C, (2)H-labeled proteins with methyl protonation in Val, Leu, and Ile (C(delta1) only) residues. Distances to methyl protons, critical for the structure determination, comprised 77 % of the long-range restraints. Initial structures were calculated on the basis of 1943 NOEs, 48 hydrogen bond and 555 dihedral angle restraints. A global pair-wise backbone rmsd of 5.5 A was obtained for these initial structures with rmsd values for the N and C domains of 2.4 and 3.8 A, respectively. Direct refinement against one-bond (1)H(N)-(15)N, (13)C(alpha)-(13)CO, (15)N-(13)CO, two-bond (1)H(N)-(13)CO and three-bond (1)H(N)-(13)C(alpha) dipolar couplings resulted in structures with large numbers of dipolar restraint violations. As an alternative to direct refinement against measured dipolar couplings we have developed an approach where discrete orientations are calculated for each peptide plane on the basis of the dipolar couplings described above. The orientation which best matches that in initial NMR structures calculated from NOE and dihedral angle restraints exclusively is used to refine further the structures using a new module written for CNS. Modeling studies from four different proteins with diverse structural motifs establishes the utility of the methodology. When applied to experimental data recorded on MBP the precision of the family of structures generated improves from 5.5 to 2.2 A, while the rmsd with respect to the X-ray structure (1dmb) is reduced from 5.1 to 3.3 A.

We have expanded our description of the energy landscape for folding of the SH3 domain of chicken alpha-spectrin by a detailed structural characterization of its denatured state ensemble (DSE). This DSE is significantly populated under mildly acidic conditions in equilibrium with the folded state. Evidence from heteronuclear nuclear magnetic resonance (NMR) experiments on (2)H, (15)N-labeled protein suggests the presence of conformers whose residual structure bears some resemblence to the structure of the folding transition state of this protein. NMR analysis in a mutant with an engineered, non-native alpha-helical tendency shows a significant amount of local non-native structure in the mutant, while the overall characteristics of the DSE are unchanged. Comparison with recent theoretical predictions of SH3 domain folding reactions reveals an interesting correlation with the predicted early events. Based on these results and recent data from other systems, we propose that the DSE of a protein will resemble the intermediate or transition state of its nearest rate-limiting step, as a consequence of simple energetic and kinetic principles.

Phosphotyrosine-binding (PTB) domains were originally identified as modular domains that recognize phosphorylated Asn-Pro-Xxx-p Tyr-containing proteins. Recent binding and structural studies of PTB domain complexes with target peptides have revealed a number of deviations from the previously described mode of interaction, with respect to both the sequences of possible targets and their structures within the complexes. This diversity of recognition by PTB domains extends and strengthens our general understanding of modular binding domain recognition.

The structures of the denatured states of the spectrin SH3 domain and a mutant designed to have a non-native helical tendency at the N terminus have been analyzed under mild acidic denaturing conditions by nuclear magnetic resonance methods with improved resolution. The wild-type denatured state has little residual structure. However, the denatured state of the mutant has an approximately 50% populated helical structure from residues 2 to 14, a region that forms part of the beta-sheet structure in the folded state. Comparison with a peptide corresponding to the same sequence shows that the helix is stabilized in the whole domain, likely by non-local interactions with other parts of the protein as suggested by changes in a region far from the mutated sequence. These results demonstrate that high populations of non-native secondary structure elements in the denatured state are compatible with the formation of the native folded structure.

The 1999 report by Mok et al. presented HN– HN NOE data recorded at 600 MHz on a highly deuterated sample of the drkN SH3 domain, a system that exists in equilibrium between a folded (Fexch) and a highly populated unfolded (Uexch) state under non-denaturing conditions. Recently the same NOE experiment has been recorded at 800 MHz (see Figure 1 legend for sample details). By comparing the two data sets it has become apparent that a portion of the previous data was misassigned. Refer to Table 1 for a summary of all current HN–HN NOE data of range (i, i þ 3) and higher. Due to the intrinsic challenges of analysing NOE data from an unfolded protein, with very low intensity or highly overlapped crosspeaks, a number of NOEs previously reported have now been attributed to spectral artifacts, while others were misassigned as long range backbone–backbone NOEs when in fact they were NOEs from backbone to nearby sidechain NH2 groups (whose resonance positions were only recently identified). The new data recorded at 800 MHz is significantly better resolved than the original experiment recorded at 600 MHz (compare Figure 1(a) versus 1(b)). Although recorded at higher field strength, the most crucial difference in the spectra was the method of processing the raw data. The F1 nitrogen dimension was linear predicted for the 600 MHz data but not the 800 MHz data, while both were linear predicted in the F2 nitrogen dimension. Successful linear prediction relies on a high signalto-noise ratio (S/N), therefore in regions of a NOESY spectrum where weak peaks exist (due to long-range or weak interactions between two atoms), linear prediction can, in some cases, make weak peaks even less intense, and conversely, make some noise look like real peaks. Close analysis of the 600 MHz data reveals that the additional linear prediction adds significant noise (as seen in Figure 1(b)), as well as “false peaks”. Use of Pf1 phage to minimise exchange-NOEs. When Pf1 filamentous phage is added to a solution sample of the drkN SH3 domain under nondenaturing buffer conditions (50 mM sodium phosphate, pH 6), the ratio of folded to unfolded conformations is significantly shifted towards the unfolded state without any measurable changes in the corresponding chemical shifts. We previously reported it is possible to stabilise the folded state by adding 0.4 M sodium sulphate to the sample, however this was the first technique we found that selectively stabilises the Uexch state. The advantage of removing folded state peaks for NOE experiments is twofold: (i) simplification of spectra from decreased overlap with Uexch peaks and (ii) elimination of the “exchange-NOE” peaks arising when the transition from one conformational state to another occurs during NOE transfer (refer to Mok et al. for a full description). While the sharpness and intensity of the peaks in spectra recorded on deuterated samples prevents the complete elimination of the folded state peaks upon addition of phage, they are significantly weaker and broader than the unfolded state peaks (see Figure 1(c)).

Cystic fibrosis is caused by mutations in the chloride channel CFTR, leading to loss of function and changes in the ion and fluid flow across epithelial surfaces. Like ABC transporters, CFTR contains two membrane spanning domains (MSDs) and two cytoplasmic nucleotide binding domains (NBDs). The formation of an NBD1/NBD2 dimer drives channel opening. The coupling helices at the base of the intracellular domains (ICLs) couple the NBDs to the MSDs of the channel. How are changes on the heterodimer interface transmitted across NBD1 to ICLs? The sensitivity of NMR spectroscopy reveals how the ICL4 binding site of NBD1 is allosterically linked to its heterodimer interface. During titrations, an ICL4 “coupling helix” peptide bound near the alpha-subdomain of NBD1, leading to destabilization and release of the C-terminal NBD1 helices 8 and 9 (H8/H9) from the heterodimer interface via an allosteric mechanism. Therefore, perturbations in one region should cause a reciprocal change in the other region. DelF508, a CF-causing mutation in the alpha-subdomain, reduces the effects of ICL4 binding on H8/H9. In contrast, DelF508-suppressor mutations, F494N and V510D, increase these effects. Helix 8 mutation, Q637R (also a DelF508-suppressor), increases the binding effects in the ICL4 binding site. Q637R also alters the dynamics in this region, suggesting that the internal motions of NBD1 are involved in transmitting changes across this plastic domain. The destabilization and release of H8/H9 from the heterodimer interface is strikingly similar to that of the regulatory extension (RE) and R region, which follow helix 9 and become more disordered and less bound to NBD1 upon phosphorylation. The RE and R region regulate NBD dimerization and, ultimately, channel opening and closing. The allosteric pathway provides insight into how dimerization may be communicated to the rest of CFTR.

SH3 is a common binding domain that mediates protein-protein interactions. The N-terminal SH3 domain of the Drosophila adaptor protein Drk is marginally stable under physiological conditions, showing nearly 50/50 equilibrium between the folded and unfolded states. Due to the dynamic ensemble nature of this unfolded state under non-denaturing conditions, the structural characterization of SH3 is difficult using ensemble measurements. Experimental data have been used to define sets of heterogeneous conformations but ensemble averaging minimizes the information content [1]. Importantly, little is known about interconversion rates within the unfolded ensemble.We performed fluorescence correlation spectroscopy (FCS) experiments on the wild-type and on a single-site mutant stabilizing the folded state. Based on this data, we estimated the size (hydrodynamic radii), of the folded (Rf), unfolded (Ru) and denatured (Rd) states. The size of unfolded state measured by FCS is in excellent agreement with the NMR-measured value, Ru/Rf = 1.30 ± 0.01 [1]. Quite surprisingly, our results indicate that the unfolded state under non-denaturing conditions is less compact than the chemically-denatured state, as Rd/Rf = 1.21 ± 0.03. This agrees with previously reported NMR and SAXS data when adequate shape factors for different conformations are considered. Using an environment sensitive dye and fitting the full correlation curve reveals a fast (∼200 ns) process within the unfolded ensemble that is absent in the folded state.Single-molecule FRET experiments were performed to quantify end-to-end distance distributions. We probed directly the conformational heterogeneity of the wild-type protein as well as the response to denaturants and osmolytes. Quite surprisingly, N- and the C-termini remained in close proximity at high denaturant concentrations for ca. 40% of the conformations, suggesting that DrkN-SH3behaves at least partially like a disordered circular chain.Reference:1. J.A. Marsh and J.D. Forman-Kay, Proteins 80(2):556-572 (2012)

Intrinsically disordered proteins (IDPs) have been implicated in the regulation of many important cellular processes, such as regulation of cell cycle progression, of transcription and of translation. IDPs are thought to function primarily in the mediation of protein-protein interactions, but detailed analysis of the molecular mechanisms that render disorder beneficial in protein interactions is required. According to a common view, IDPs are usually thought to undergo disorder-to-order transitions upon interaction with their binding partners. It is becoming increasingly clear that predominantly disordered protein complexes are functionally relevant. We have shown previously that the intrinsically disordered cyclin dependent kinase (CDK) inhibitor Sic1 interacts with the substrate adapter, Cdc4, of its ubiquitin ligase via multiple phosphorylated binding motifs in a dynamic complex. Cdc4 is the substrate recognition subunit of a culin ubiquitin ligase and targets Sic1 for degradation at the G1/S phase transition of the yeast cell cycle. Individual binding motifs in Sic1 are ordered transiently without a global disorder-to-order transition upon binding Cdc4. The dynamic complex allows for engagement of several phosphorylation sites in a dynamic interface resulting in an affinity that depends on the number of phosphorylation sites in an ultrasensitive manner. The dynamic nature of the complex allows for ‘counting’ of phosphorylation sites via largely electrostatic interactions. The Sic1-Cdc4 interaction therefore acts as a sensor of the concentration of active kinase and the cell cycle status. We continue to use NMR spectroscopy and other biophysical methods to study dynamic interactions in the ubiquitin proteasome pathway with the objective of unraveling nature's repertoire of disorder in protein function. The combination of disorder with multi-site phosphorylation may serve as a general means to set thresholds in regulated protein-protein interactions.

The SH3 domain is a commonly found modular binding domain that functions to mediate protein-protein interactions and has been a frequent target of protein folding and other biophysical studies. The N-terminal SH3 domain (6.8 kDa) of the Drosophila adapter protein Drk is marginally stable under physiological conditions and exists in nearly 50/50 equilibrium between the folded and unfolded states, with a slow conversion rate of 2.2 s−1(1). Due to the dynamic ensemble nature of this unfolded state under non-denaturing conditions it is difficult to characterize structurally. Experimental data have been used to define sets of heterogeneous conformations but ensemble averaging minimizes the information content (2). Importantly, little is known about inter-conversion rates within the unfolded ensemble.Fluorescence correlation spectroscopy (FCS) experiments performed on the wild-type and on a mutant that stabilizes the folded state (T22G) provided the hydrodynamic radii of the folded (Rh,f), unfolded (Rh,u) and denatured (Rh,d) states at 20°C. Quite surprisingly, the results indicate that the unfolded state under non-denaturing conditions is less compact than the chemically-denatured state (Rh,u/Rh,f=1.31±0.05 vs. Rh,d/Rh,f=1.21±0.03). However, this can be explained by introducing adequate shape factors in previously reported NMR/SAXS data. In addition the FCS measured compactness of unfolded state is in an excellent agreement with PFG-NMR value of Rh,u/Rh,f=1.30±0.01 (2). Furthermore, FCS analysis reveals a fast 200 ns process within the unfolded ensemble. To resolve slow (milli)second conformational dynamics, dual cysteine mutant (E2C-60C) was labelled with the Bodipy Fl/Alexa 647 FRET pair and single proteins were encapsulated in surface-tethered liposomes for long observations. This study provides further insights into the unfolded state heterogeneity and its importance in protein folding in general.1.M Tollinger et al., J. Am. Chem. Soc.;123(46):11341-11352 (2001).2.WY Choy et al., J. Mol. Biol.;316(1):101-112 (2002).