FF Domain Research Articles

Backbone 1H, 15N, and 13C resonance assignments of the FF1 domain from P190A RhoGAP in 5 and 8M urea.

The Rho GTPase (Ras homolog GTPases) system is a crucial signal transducer that regulates various cellular processes, including cell cycle and migration, genetic transcription, and apoptosis. In this study, we investigated the unfolded state of the first FF domain (FF1) of P190A RhoGAP, which features four tandem FF domains. For signal transduction, FF1 is phosphorylated at tyrosine 308 (Y308), which is buried in the hydrophobic core and is inaccessible to kinases in the folded domain. It was proposed, therefore, that the phosphorylation occurs in a transiently populated unfolded state of FF1. To probe the folding pathway of the RhoGAP FF1 domain, here we have performed a nearly complete backbone resonance assignments of a putative partially unfolded state of FF1 in 5M urea and its fully unfolded state in 8M urea.

Increasing the accuracy of exchange parameters reporting on slow dynamics by performing CEST experiments with ‘high’ B1 fields

Over the last decade chemical exchange saturation transfer (CEST) NMR methods have emerged as powerful tools to characterize biomolecular conformational dynamics occurring between a visible major state and ‘invisible’ minor states. The ability of the CEST experiment to detect these minor states, and provide precise exchange parameters, hinges on using appropriate B1 field strengths during the saturation period. Typically, a pair of B1 fields with ω1 (=2πB1) values around the exchange rate kex are chosen. Here we show that the transverse relaxation rate of the minor state resonance (R2,B) also plays a crucial role in determining the B1 fields that lead to the most informative datasets. Using K=kexkex+R2,B12 ≥ kex, to guide the choice of B1, instead of kex, leads to data wherefrom substantially more accurate exchange parameters can be derived. The need for higher B1 fields, guided by K, is demonstrated by studying the conformational exchange in two mutants of the 71 residue FF domain with kex ∼ 11 s−1 and ∼ 72 s−1, respectively. In both cases analysis of CEST datasets recorded using B1 field values guided by kex lead to imprecise exchange parameters, whereas using B1 values guided by K resulted in precise site-specific exchange parameters. The conclusions presented here will be valuable while using CEST to study slow processes at sites with large intrinsic relaxation rates, including carbonyl sites in small to medium sized proteins, amide 15N sites in large proteins and when the minor state dips are broadened due to exchange among the minor states.

The A39G FF domain folds on a volcano-shaped free energy surface via separate pathways

The A39G FF domain folds on a volcano-shaped free energy surface via separate pathways

Beyond slow two-state protein conformational exchange using CEST: applications to three-state protein interconversion on the millisecond timescale.

Although NMR spectroscopy is routinely used to study the conformational dynamics of biomolecules, robust analyses of the data are challenged in cases where exchange is more complex than two-state, such as when a 'visible' major conformer exchanges with two 'invisible' minor states on the millisecond timescale. It is becoming increasingly clear that chemical exchange saturation transfer (CEST) NMR experiments that were initially developed to study systems undergoing slow interconversion are also sensitive to intermediate-fast timescale biomolecular conformational exchange. Here we investigate the utility of the amide 15N CEST experiment to characterise protein three-state exchange occurring on the millisecond timescale by studying the interconversion between the folded (F) state of the FF domain from human HYPA/FBP11 (WT FF) and two of its folding intermediates I1 and I2. Although 15N CPMG experiments are consistent with the F state interconverting with a single minor state on the millisecond timescale, 15N CEST data clearly establish an exchange process between F and a pair of minor states. A unique three-state exchange model cannot be obtained by analysis of 15N CEST data recorded at a single temperature. However, including the relative sign of the difference in the chemical shifts of the two minor states based on a simple two-state analysis of CEST data recorded at multiple temperatures, results in a robust three-state model in which the F, I1 and I2 states interconvert with each other on the millisecond timescale ( ~ 550s-1, ~ 1200s-1, ~ 5000s-1), with I1 and I2 sparsely populated at ~ 0.15% and ~ 0.35%, respectively, at 15°C. A computationally demanding grid-search of exchange parameter space is not required to extract the best-fit exchange parameters from the CEST data. The utility of the CEST experiment, thus, extends well beyond studies of conformers in slow exchange on the NMR chemical shift timescale, to include systems with interconversion rates on the order of thousands/second.

Binding by calmodulin is coupled to transient unfolding of the third FF domain of Prp40A.

Human pre-mRNA processing protein 40 homolog A (hPrp40A) is a splicing factor that interacts with the Huntington's disease protein huntingtin (Htt). Evidence has accumulated that both Htt and hPrp40A are modulated by the intracellular Ca2+ sensor calmodulin (CaM). Here we report characterization of the interaction of human CM with the third FF domain (FF3 ) of hPrp40A using calorimetric, fluorescence and structural approaches. Homology modeling, differential scanning calorimetry and small angle X-ray scattering (SAXS) data show FF3 forms a folded globular domain. CaM was found to bind FF3 in a Ca2+ -dependent manner with a 1:1 stoichiometry and a dissociation constant (Kd ) of 25 ± 3μM at 25°C. NMR studies showed that both domains of CaM are engaged in binding and SAXS analysis of the FF3 -CaM complex revealed CaM occupies an extended configuration. Analysis of the FF3 sequence showed that the anchors for CaM binding must be buried in its hydrophobic core, suggesting that binding to CaM requires unfolding of FF3 . Trp anchors were proposed based on sequence analysis and confirmed by intrinsic Trp fluorescence of FF3 upon binding of CaM and substantial reductions in affinity for Trp-Ala FF3 mutants. The consensus model of the complex showed that binding to CaM binding occurs to an extended, non-globular state of the FF3 , consistent with coupling to transient unfolding of the domain. The implications of these results are discussed in the context of the complex interplay of Ca2+ signaling and Ca2+ sensor proteins in modulating Prp40A-Htt function.

The A39G FF domain folds on a volcano-shaped free energy surface via separate pathways

Conformational dynamics play critical roles in protein folding, misfolding, function, misfunction, and aggregation. While detecting and studying the different conformational states populated by protein molecules on their free energy surfaces (FESs) remain a challenge, NMR spectroscopy has emerged as an invaluable experimental tool to explore the FES of a protein, as conformational dynamics can be probed at atomic resolution over a wide range of timescales. Here, we use chemical exchange saturation transfer (CEST) to detect "invisible" minor states on the energy landscape of the A39G mutant FF domain that exhibited "two-state" folding kinetics in traditional experiments. Although CEST has mostly been limited to studies of processes with rates between ∼5 to 300 s-1 involving sparse states with populations as low as ∼1%, we show that the line broadening that is often associated with minor state dips in CEST profiles can be exploited to inform on additional conformers, with lifetimes an order of magnitude shorter and populations close to 10-fold smaller than what typically is characterized. Our analysis of CEST profiles that exploits the minor state linewidths of the 71-residue A39G FF domain establishes a folding mechanism that can be described in terms of a four-state exchange process between interconverting states spanning over two orders of magnitude in timescale from ∼100 to ∼15,000 μs. A similar folding scheme is established for the wild-type domain as well. The study shows that the folding of this small domain proceeds through a pair of sparse, partially structured intermediates via two discrete pathways on a volcano-shaped FES.

Meta Reinforcement Learning for Heuristic Planing

Heuristic planning has a central role in classical planning applications and competitions. Thanks to this success, there has been an increasing interest in using Deep Learning to create high-quality heuristics in a supervised fashion, learning from optimal solutions of previously solved planning problems. Meta-Reinforcement learning is a fast growing research area concerned with learning, from many tasks, behaviours that can quickly generalize to new tasks from the same distribution of the training ones. We make a connection between meta-reinforcement learning and heuristic planning, showing that heuristic functions meta-learned from planning problems, in a given domain, can outperform both popular domain-independent heuristics, and heuristics learned by supervised learning. Furthermore, while most supervised learning algorithms rely on ad-hoc encodings of the state representation, our method uses as input a general PDDL 3.1 description. We evaluated our heuristic with an A* planner on six domains from the International Planning Competition and the FF Domain Collection, showing that the meta-learned heuristic leads to the expansion, on average, of fewer states than three popular heuristics used by the FastDownward planner, and a supervised-learned heuristic.

In situ AFM investigation of dual-mode self-assembling peptide

Nanostructures/patterns formed by biomolecules can produce different physicochemical properties in terms of hydrophobicity, zeta-potential, color, etc., which play paramount roles in life. Peptides, as the main bio-building blocks, can form nanostructures with different functions, either in solutions or on interfaces. Previously, we synthesized a short peptide with the inspiration of an Alzheimer’s disease-related peptide: amyloid β peptide (A-β), namely GAV-9, which can epitaxially self-assemble into regular nanofilaments on liquid–solid interfaces, and it was found that both the hydrophobicity and charge state of the interfaces can significantly influence its assembling behavior. It was also reported that another A-β-containing dipeptide, FF, can self-assemble into nanostructures in solutions. Owing to the close relationship between these two short peptides, it is interesting to conjugate them into a de novo peptide with two separated structural domains and study its self-assembling behavior. To this end, herein we have synthesized the GAV-FF peptide with a sequence of NH2-VGGAVVAGVFF-CONH2 and verified its self-assembling property using the in situ liquid-phase atomic force microscopy. The results show that the GAV-FF peptide can self-assemble into nanofilaments both in solutions and on aqueous–solid interfaces, but with different morphologies. The FF domain accelerates the template-assisted self-assembling (TASA) process of the GAV domain, which in return enhances the solubility of FF in aqueous solutions and further participates in the fibrillization of FF. The current results could help deepen the understanding of the aggregation mechanism of disease-related peptides and could also shed light on the strategies to create artificial bio-functional nanostructures/patterns, which hold a significant potential for biomedical applications.

Global Protein Stabilization Does Not Suffice to Prevent Amyloid Fibril Formation.

Mutations or cellular conditions that destabilize the native protein conformation promote the population of partially unfolded conformations, which in many cases assemble into insoluble amyloid fibrils, a process associated with multiple human pathologies. Therefore, stabilization of protein structures is seen as an efficient way to prevent misfolding and subsequent aggregation. This has been suggested to be the underlying reason why proteins living in harsh environments, such as the extracellular space, have evolved disulfide bonds. The effect of protein disulfides on the thermodynamics and kinetics of folding has been extensively studied, but much less is known on its effect on aggregation reactions. Here, we designed a single point mutation that introduces a disulfide bond in the all-α FF domain, a protein that, despite being devoid of preformed β-sheets, forms β-sheet-rich amyloid fibrils. The novel and unique covalent bond in the FF domain dramatically increases its thermodynamic stability and folding speed. Nevertheless, these optimized properties cannot counteract the inherent aggregation propensity of the protein, thus indicating that a high global protein stabilization does not suffice to prevent amyloid formation unless it contributes to hide from exposure the specific regions that nucleate the aggregation reaction.

Molecular biophysical characterization of the third FF domain of Homo sapiens Prp40 homolog A

Homo sapiens Prp40 Homolog A (HsPrp40A) is a nuclear protein that is involved in pre-mRNA splicing. This modular protein has two WW domains followed by six tandem FF domains. Previous studies demonstrate that the WW domain-mediated interactions of HsPrp40A are implicated in genetic disorders such as Huntington's disease and Rett syndrome. However, little is known about the biological implications related to HsPrp40A's FF domains. Recently, we determined that HsPrp40A peptide interacts with high affinity with a calcium-binding protein known as centrin using isothermal titration calorimetry and two-dimensional infrared correlation spectroscopy. Interestingly, this centrin-binding site is located within the third FF (FF3) domain of HsPrp40A. Herein, we present a comparative molecular biophysical study using circular dichroism and two-dimensional infrared correlation spectroscopy to explore the molecular behavior of HsPrp40A's FF3 domain under thermal stress. These results may provide new insights about the stability of HsPrp40A's FF3 domain.

Interaction of p190A RhoGAP with eIF3A and Other Translation Preinitiation Factors Suggests a Role in Protein Biosynthesis

The negative regulator of Rho family GTPases, p190A RhoGAP, is one of six mammalian proteins harboring so-called FF motifs. To explore the function of these and other p190A segments, we identified interacting proteins by tandem mass spectrometry. Here we report that endogenous human p190A, but not its 50% identical p190B paralog, associates with all 13 eIF3 subunits and several other translational preinitiation factors. The interaction involves the first FF motif of p190A and the winged helix/PCI domain of eIF3A, is enhanced by serum stimulation and reduced by phosphatase treatment. The p190A/eIF3A interaction is unaffected by mutating phosphorylated p190A-Tyr308, but disrupted by a S296A mutation, targeting the only other known phosphorylated residue in the first FF domain. The p190A-eIF3 complex is distinct from eIF3 complexes containing S6K1 or mammalian target of rapamycin (mTOR), and appears to represent an incomplete preinitiation complex lacking several subunits. Based on these findings we propose that p190A may affect protein translation by controlling the assembly of functional preinitiation complexes. Whether such a role helps to explain why, unique among the large family of RhoGAPs, p190A exhibits a significantly increased mutation rate in cancer remains to be determined.

Detect Fast-Flux Domains Through Response Time Differences

A fast-flux service network (FFSN) uses dynamic DNS to map a dynamic domain, called fast-flux domain (FF domain), to various IP addresses and uses flux bots to redirect network traffic. Due to its powerful capability to conceal the hosts hidden behind the flux bots, FFSNs are widely adopted by attackers to cover various scams. Although diverse promising solutions have been proposed to detect FF domains, they face the same problem-different countermeasures could be used to bypass their detection. Hence, it becomes a critical issue to develop a new detection solution. According to our survey, unlike normal network services that use dynamic DNS to balance the workloads of their hosts, FFSNs utilize dynamic DNS to hide important bots. As a result, the response time of subsequent requests to an FF domain becomes more fluctuating. Based on the response time differences, this paper develops a new metric, Fast-Flux Score (FF-Score), to detect FF domains. Our system, called fast-flux domain detector (FFDD), is used on a computer that could be an end host or an IDS. A user with a set of unknown URLs, which may be obtained from spam or social networks, can simply determine whether they are benign domains or fast-flux ones using FFDD. Experimental results show that FFDD can accurately detect FF domains with only a 0.3% false positive rate and a 2% false negative rate. It takes less than 20 min for FFDD to determine whether a domain is an FF domain. In addition, FFDD is a lightweight stand-alone system; hence, it does not require special support from an ISP or any other network service.

A Similar In Vitro and In Cell Lysate Folding Intermediate for the FF Domain

Understanding the mechanisms by which proteins fold into their three-dimensional structures, including a description of the intermediates that are formed during the folding process, remains a goal of protein science. Most studies are performed under carefully controlled conditions in which the folding reaction is monitored in a buffer solution that is far from the natural milieu of the cell. Here, we have used 13C and 1H relaxation dispersion NMR spectroscopy to study folding of the FF domain in both Escherichia coli and Saccharomyces cerevisiae cellular lysates. We find that a conformationally excited state is populated in both lysates, which is very similar in structure to a folding intermediate observed in previous studies in buffer, with the kinetics and thermodynamics of the interconversion between native and intermediate conformers somewhat changed. The results point to the importance of extending folding studies beyond the test tube yet emphasize that insights can be obtained through careful experiments recorded in controlled buffer solutions.

Viscosity-Dependent Kinetics of Protein Conformational Exchange: Microviscosity Effects and the Need for a Small Viscogen

Conformational rearrangements are critical to a variety of biological processes including protein folding and misfolding, ligand binding, enzyme catalysis, and signal transduction. Viscosity-dependent kinetics measurements can provide crucial insights into the dynamics of protein conformational exchange by highlighting the relative importance of frictional forces derived from either solvent or from internal protein interactions in activating the exchange reaction. Here, we analyze the kinetics of interconversion between the native and intermediate states of the four helix bundle FF domain recorded in solutions containing the viscogens glycerol or bovine serum albumin (BSA), using the viscosity measured from the translational diffusion of probes of different sizes. In the large viscogen BSA, we demonstrate that vastly different internal friction values are obtained using the different viscosity measures, leading to conflicting interpretations of the role of solvent friction in the interconversion. We show that this can be a consequence of the small effective hydrodynamic radius of the protein conformational transition and differences between solution micro- and macroscopic viscosities that are germane in this case. In general, correct values of internal friction can only be obtained by carrying out measurements using small viscogens.

Human p190A RhoGAP binds eIF3A and exists in a complex with the 48S translational pre‐initiation complex (1013.8)

By regulating the actin cytoskeleton Rho GTPases control biological processes such as cell migration, adhesion and polarity. p190A RhoGAP is an important yet poorly understood Rho GTPase regulator. To shed further light on the functions of p190A RhoGAP, we performed mass spectroscopic analysis of associated proteins. Here we report that human p190A, but not its related paralog p190B, forms a stable complex with multiple subunits of the 48S translational pre‐initiation complex. Complex formation involves a direct interaction between the first FF domain of p190A and the PCI/PAM domain of eIF3A. This interaction does not appear to be regulated by Y308 phosphorylation, previously found to control the p190A directed cytoplasmic sequestration of TFII‐I, but may instead by controlled by phosphorylation of S296 by an as yet unknown protein kinase. Although other reasons for the detected interaction cannot be ruled out, we speculate that p190A RhoGAP may serve as an anchoring protein allowing the localized translation of specific mRNAs.Grant Funding Source: RO1 GM084220

Visualizing Side Chains of Invisible Protein Conformers by Solution NMR

Sparsely populated and transiently formed protein conformers can play key roles in many biochemical processes. Understanding the structure function paradigm requires, therefore, an atomic-resolution description of these rare states. However, they are difficult to study because they cannot be observed using standard biophysical techniques. In the past decade, NMR methods have been developed for structural studies of these elusive conformers, focusing primarily on backbone 1H, 15N and 13C nuclei. Here we extend the methodology to include side chains by developing a 13C-based chemical exchange saturation transfer experiment for the assignment of side-chain aliphatic 13C chemical shifts in uniformly 13C labeled proteins. A pair of applications is provided, involving the folding of β-sheet Fyn SH3 and α-helical FF domains. Over 96% and 89% of the side-chain 13C chemical shifts for excited states corresponding to the unfolded conformation of the Fyn SH3 domain and a folding intermediate of the FF domain, respectively, have been obtained, providing insight into side-chain packing and dynamics.

Trifluoroethanol Modulates Amyloid Formation by the All α-Helical URN1 FF Domain

Amyloid fibril formation is implicated in different human diseases. The transition between native α-helices and nonnative intermolecular β-sheets has been suggested to be a trigger of fibrillation in different conformational diseases. The FF domain of the URN1 splicing factor (URN1-FF) is a small all-α protein that populates a molten globule (MG) at low pH. Despite the fact that this conformation maintains most of the domain native secondary structure, it progressively converts into β-sheet enriched and highly ordered amyloid fibrils. In this study, we investigated if 2,2,2-trifluoroethanol (TFE) induced conformational changes that affect URN1-FF amyloid formation. Despite TFE having been shown to induce or increase the aggregation of both globular and disordered proteins at moderate concentrations, we demonstrate here that in the case of URN1-FF it reinforces its intrinsic α-helical structure, which competes the formation of aggregated assemblies. In addition, we show that TFE induces conformational diversity in URN1-FF fibrils, in such a way that the fibrils formed in the presence and absence of the cosolvent represent different polymorphs. It is suggested that the effect of TFE on both the soluble and aggregated states of URN1-FF depends on its ability to facilitate hydrogen bonding.

Defining a length scale for millisecond-timescale protein conformational exchange.

Although atomic resolution 3D structures of protein native states and some folding intermediates are available, the mechanism of interconversion between such states remains poorly understood. Here we study the four-helix bundle FF module, which folds via a transiently formed and sparsely populated compact on-pathway intermediate, I. Relaxation dispersion NMR spectroscopy has previously been used to elucidate the 3D structure of this intermediate and to establish that the conformational exchange between the I and the native, N, states of the FF domain is driven predominantly by water dynamics. In the present study we use NMR methods to define a length scale for the FF I-N transition, namely the effective hydrodynamic radius (EHR) that provides an average measure of the size of the structural units participating in the transition at any given time. Our experiments establish that the EHR is less than 4 Å, on the order of the size of one to two amino acid side chains, much smaller than the FF domain hydrodynamic radius (13 Å). The small magnitude of the EHR provides strong evidence that the I-N interconversion does not proceed via the synchronous motion of large clusters of amino acid residues, but rather by the exposure/burial of one or two side chains from solvent at any given time. Because the hydration of small hydrophobic solutes (< 4 Å) does not involve considerable dewetting or disruption of the water-hydrogen bonding network, the FF domain I-N transition does not require appreciable changes to the structure of the surrounding water.

Specific Interaction of the Transcription Elongation Regulator TCERG1 with RNA Polymerase II Requires Simultaneous Phosphorylation at Ser2, Ser5, and Ser7 within the Carboxyl-terminal Domain Repeat

The human transcription elongation regulator TCERG1 physically couples transcription elongation and splicing events by interacting with splicing factors through its N-terminal WW domains and the hyperphosphorylated C-terminal domain (CTD) of RNA polymerase II through its C-terminal FF domains. Here, we report biochemical and structural characterization of the C-terminal three FF domains (FF4-6) of TCERG1, revealing a rigid integral domain structure of the tandem FF repeat that interacts with the hyperphosphorylated CTD (PCTD). Although FF4 and FF5 adopt a classical FF domain fold containing three orthogonally packed α helices and a 310 helix, FF6 contains an additional insertion helix between α1 and α2. The formation of the integral tandem FF4-6 repeat is achieved by merging the last helix of the preceding FF domain and the first helix of the following FF domain and by direct interactions between neighboring FF domains. Using peptide column binding assays and NMR titrations, we show that binding of the FF4-6 tandem repeat to the PCTD requires simultaneous phosphorylation at Ser(2), Ser(5), and Ser(7) positions within two consecutive Y(1)S(2)P(3)T(4)S(5)P(6)S(7) heptad repeats. Such a sequence-specific PCTD recognition is achieved through CTD-docking sites on FF4 and FF5 of TCERG1 but not FF6. Our study presents the first example of a nuclear factor requiring all three phospho-Ser marks within the heptad repeat of the CTD for high affinity binding and provides a molecular interpretation for the biochemical connection between the Ser(7) phosphorylation enrichment in the CTD of the transcribing RNA polymerase II over introns and co-transcriptional splicing events.

The N-terminal Helix Controls the Transition between the Soluble and Amyloid States of an FF Domain

BackgroundProtein aggregation is linked to the onset of an increasing number of human nonneuropathic (either localized or systemic) and neurodegenerative disorders. In particular, misfolding of native α-helical structures and their self-assembly into nonnative intermolecular β-sheets has been proposed to trigger amyloid fibril formation in Alzheimer’s and Parkinson’s diseases.MethodsHere, we use a battery of biophysical techniques to elucidate the conformational conversion of native α-helices into amyloid fibrils using an all-α FF domain as a model system.ResultsWe show that under mild denaturing conditions at low pH this FF domain self-assembles into amyloid fibrils. Theoretical and experimental dissection of the secondary structure elements in this domain indicates that the helix 1 at the N-terminus has both the highest α-helical and amyloid propensities, controlling the transition between soluble and aggregated states of the protein.ConclusionsThe data illustrates the overlap between the propensity to form native α-helices and amyloid structures in protein segments.SignificanceThe results presented contribute to explain why proteins cannot avoid the presence of aggregation-prone regions and indeed use stable α-helices as a strategy to neutralize such potentially deleterious stretches.