Global Fold Research Articles

Three-Dimensional Protein Structure Determination Using Pseudocontact Shifts of Backbone Amide Protons Generated by Double-Histidine Co2+-Binding Motifs at Multiple Sites.

Pseudocontact shifts (PCSs) generated by paramagnetic metal ions contribute highly informative long-range structure restraints that can be measured in solution and are ideally suited to guide structure prediction algorithms in determining global protein folds. We recently demonstrated that PCSs, which are relatively small but of high quality, can be generated by a double-histidine (dHis) motif in an α-helix, which provides a well-defined binding site for a single Co2+ ion. Here we show that PCSs of backbone amide protons generated by dHis-Co2+ motifs positioned in four different α-helices of a protein deliver excellent restraints to determine the three-dimensional (3D) structure of a protein in a way akin to the global positioning system (GPS). We demonstrate the approach with GPS-Rosetta calculations of the 3D structure of the C-terminal domain of the chaperone ERp29 (ERp29-C). Despite the relatively small size of the PCSs generated by the dHis-Co2+ motifs, the structure calculations converged readily. Generating PCSs by the dHis-Co2+ motif thus presents an excellent alternative to the use of lanthanide tags.

Characterization of human frataxin missense variants in cancer tissues.

Human frataxin is an iron-binding protein involved in the mitochondrial iron-sulfur (Fe-S) clusters assembly, a process fundamental for the functional activity of mitochondrial proteins. Decreased level of frataxin expression is associated with the neurodegenerative disease Friedreich ataxia. Defective function of frataxin may cause defects in mitochondria, leading to increased tumorigenesis. Tumor-initiating cells show higher iron uptake, a decrease in iron storage and a reduced Fe-S clusters synthesis and utilization. In this study, we selected, from COSMIC database, the somatic human frataxin missense variants found in cancer tissues p.D104G, p.A107V, p.F109L, p.Y123S, p.S161I, p.W173C, p.S181F, and p.S202F to analyze the effect of the single amino acid substitutions on frataxin structure, function, and stability. The spectral properties, the thermodynamic and the kinetic stability, as well as the molecular dynamics of the frataxin missense variants found in cancer tissues point to local changes confined to the environment of the mutated residues. The global fold of the variants is not altered by the amino acid substitutions; however, some of the variants show a decreased stability and a decreased functional activity in comparison with that of the wild-type protein.

Sequence-specific dynamic information in proteins.

We examine the local and global properties of the average B-factor, 〈B〉, as a residue-specific indicator of protein dynamic characteristics. It has been shown that values of 〈B〉 for the 20 amino acids differ in a statistically significant manner, and that, while strongly determined by the static physical properties of amino acids, they also encode averaged information about the influence of global fold on single-residue dynamics. Therefore, complete sequences of amino acids also encode fold-related global dynamic information, in addition to the local information that arises from static physical properties. We show that the relative magnitudes of these two contributions can be determined using Fourier methods, which represent the global properties of the sequences. It has also been shown that the behavior of Fourier components of 〈B〉 differs, with very high statistical significance, between structural groups, and that this information is not available from a comparable analysis of static amino acid properties.

A two-dimensional replica-exchange molecular dynamics method for simulating RNA folding using sparse experimental restraints.

We present a 2D replica exchange protocol incorporating secondary structure information to dramatically improve 3D RNA folding using molecular dynamics simulations. We show that incorporating base-pairing restraints into all-atom, explicit solvent simulations enables the accurate recapitulation of the global tertiary fold for 4 representative RNAs ranging in length from 24 to 68 nt. This method can potentially utilize base-pairing information from a wide variety of experimental inputs to predict complex RNA tertiary folds including pseudoknots, multi-loop junctions, and non-canonical interactions.

Translation-coupled protein folding assay using a protease to monitor the folding status.

Protein folding is an essential prerequisite for proteins to execute nearly all cellular functions. There is a growing demand for a simple and robust method to investigate protein folding on a large-scale under the same conditions. We previously developed a global folding assay system, in which proteins translated using an Escherichia coli-based cell-free translation system are centrifuged to quantitate the supernatant fractions. Although the assay is based on the assumption that the supernatants contain the folded native states, the supernatants also include nonnative unstructured proteins. In general, proteases recognize and degrade unstructured proteins, and thus we used a protease to digest the unstructured regions to monitor the folding status. The addition of Lon protease during the translation of proteins unmasked subfractions, not only in the soluble fractions but also in the aggregation-prone fractions. We translated ∼90 E. coli proteins in the protease-inclusion assay, in the absence and presence of chaperones. The folding assay, which sheds light on the molecular mechanisms underlying the aggregate formation and the chaperone effects, can be applied to a large-scale analysis.

Why protein conformers in molecular dynamics simulations differ from theircrystal structures: a thermodynamic insight

Conformers generally deviate structurally from their starting X-ray crystal structures early in molecular dynamics (MD) simulations. Studies have recognized such structural differences and attempted to provide an explanation for and justify the necessity of MD equilibrations. However, a detailed explanation based on fundamental physics and validation on a large ensemble of protein structures is still missing. Here we provide the first thermodynamic insights into the radically different thermodynamic conditions of crystallization solutions and conventional MD simulations. Crystallization solution conditions can lead to nonphysiologically high ion concentrations, low temperatures, and crystal packing with strong specific protein--protein interactions, not present under physiological conditions. These differences affect protein conformations and functions, and MD structures equilibrated or simulated under physiological conditions are usually expected to differ from their X-ray structures at a local scale, while the global fold is usually maintained. To quantify this property, we performed conventional MD simulations for over 70 different proteins spanning a broad range of molecular size and structural and functional families. Our analysis shows that crystal structures are good starting points; however, they do not represent structures in their physiological environment. This fact has to be taken into consideration when computational methods dependent on atomic coordinates, such as substrate/ligand docking, are used to guide experimental analyses.

Manipulating DNA G-Quadruplex Structures by Using Guanosine Analogues.

The ability to control the folding topology of DNA G-quadruplexes allows for rational design of quadruplex-based scaffolds for potential use in various therapeutic and technological applications. By exploiting the distinct conformational properties of some base- and sugar-modified guanosine surrogates, conformational transitions can be induced through their judicious incorporation at specific sites in the quadruplex core. Changes may involve tetrad polarity inversions with conservation of the global fold or complete refolding to new topologies. Reliable predictions relating to low-energy conformers formed upon specific chemical perturbations of the system and the rational design of modified sequences suffer from our still limited understanding of the subtle interplay of various favorable and unfavorable interactions within a particular quadruplex scaffold. However, aided by an increasing number of systematic substitution experiments and high-resolution structures of modified quadruplex variants, critical interactions, in addition to glycosidic bond angle propensities, are starting to emerge as important contributors to modification-driven quadruplex refolding.

CLC Conformational Landscape as Studied by SMFRET

CLC secondary active transporters catalyze the exchange of Cl− for H+ ions across the cellular membrane. The available CLC crystal structures all share the same global protein conformational fold, which suggests that the transport mechanism may involve only movements of a conserved glutamate residue that has been observed to adopt different side-chain rotamers. However, NMR and DEER measurements have revealed additional motions throughout the CLC protein, including in a region 20 Å away from this glutamate site. Cross-linking to restrict movement at this latter region inhibits transport, thus connecting the protein conformational dynamics to its function. To investigate further the CLC conformational landscape, we used single-molecule FRET (smFRET) to investigate substrate (H+) induced protein conformational changes. These smFRET studies show that there are H+-dependent conformational changes at both the extracellular and intracellular sides of the protein. The relevance of these changes to the H+ transport mechanism is supported by their absence in a mutant that transports Cl− but not H+. To better understand these changes, triangulation experiments are being designed and performed to obtain distance constraints at both the extracellular and intracellular sides.

Allosteres to regulate neurotransmitter sulfonation

Catecholamine neurotransmitter levels in the synapses of the brain shape human disposition-cognitive flexibility, aggression, depression, and reward seeking-and manipulating these levels is a major objective of the pharmaceutical industry. Certain neurotransmitters are extensively sulfonated and inactivated by human sulfotransferase 1A3 (SULT1A3). To our knowledge, sulfonation as a therapeutic means of regulating transmitter activity has not been explored. Here, we describe the discovery of a SULT1A3 allosteric site that can be used to inhibit the enzyme. The structure of the new site is determined using spin-label-triangulation NMR. The site forms a cleft at the edge of a conserved ∼30-residue active-site cap that must open and close during the catalytic cycle. Allosteres anchor into the site via π-stacking interactions with two residues that sandwich the planar core of the allostere and inhibit the enzyme through cap-stabilizing interactions with substituents attached to the core. Changes in cap free energy were calculated ab initio as a function of core substituents and used to design and synthesize a series of inhibitors intended to progressively stabilize the cap and slow turnover. The inhibitors bound tightly (34 nm to 7.4 μm) and exhibited progressive inhibition. The cap-stabilizing effects of the inhibitors were experimentally determined and agreed remarkably well with the theoretical predictions. These studies establish a reliable heuristic for the design of SULT1A3 allosteric inhibitors and demonstrate that the free-energy changes of a small, dynamic loop that is critical for SULT substrate selection and turnover can be calculated accurately.

Solution structure of TbTFIIS2-2 PWWP domain from Trypanosoma brucei and its binding to H4K17me3 and H3K32me3.

Posttranslational modifications (PTMs) of core histones, such as histone methylation, play critical roles in a variety of biological processes including transcription regulation, chromatin condensation and DNA repair. In T. brucei, no domain recognizing methylated histone has been identified so far. TbTFIIS2-2, as a potential transcription elongation factors in T. brucei, contains a PWWP domain in the N-terminus which shares low sequence similarity compared with other PWWP domains and is absent from other TFIIS factors. In the present study, the solution structure of TbTFIIS2-2 PWWP domain was determined by NMR spectroscopy. TbTFIIS2-2 PWWP domain adopts a global fold containing a five-strand β-barrel and two C-terminal α-helices similar to other PWWP domains. Moreover, through systematic screening, we revealed that TbTFIIS2-2 PWWP domain is able to bind H4K17me3 and H3K32me3. Meanwhile, we identified the critical residues responsible for the binding ability of TbTFIIS2-2 PWWP domain. The conserved cage formed by the aromatic amino acids in TbTFIIS2-2 PWWP domain is essential for its binding to methylated histones.

Two-State Folding of the Outer Membrane Protein X into a Lipid Bilayer Membrane.

Folding and insertion of β-barrel membrane proteins into native membranes is efficiently catalyzed by β-barrel assembly machineries. Understanding this catalysis requires a detailed description of the corresponding uncatalyzed folding mechanisms, which however have so far remained largely unclear. Herein, the folding and membrane insertion of the E. coli outer membrane protein X (OmpX) into 1,2-didecanoyl-sn-glycero-3-phosphocholine (PC10:0) membranes is resolved at the atomic level. By combining four different experimental techniques, global folding kinetics were correlated with global and local hydrogen bond-formation kinetics. Under a well-defined reaction condition, these processes follow single-exponential velocity laws, with rate constants identical within experimental error. The data thus establish, at atomic resolution, that OmpX folds and inserts into the lipid bilayer of PC10:0 liposomes by a two-state mechanism.

Two‐State Folding of the Outer Membrane Protein X into a Lipid Bilayer Membrane

AbstractFolding and insertion of β‐barrel membrane proteins into native membranes is efficiently catalyzed by β‐barrel assembly machineries. Understanding this catalysis requires a detailed description of the corresponding uncatalyzed folding mechanisms, which however have so far remained largely unclear. Herein, the folding and membrane insertion of the E. coli outer membrane protein X (OmpX) into 1,2‐didecanoyl‐sn‐glycero‐3‐phosphocholine (PC10:0) membranes is resolved at the atomic level. By combining four different experimental techniques, global folding kinetics were correlated with global and local hydrogen bond‐formation kinetics. Under a well‐defined reaction condition, these processes follow single‐exponential velocity laws, with rate constants identical within experimental error. The data thus establish, at atomic resolution, that OmpX folds and inserts into the lipid bilayer of PC10:0 liposomes by a two‐state mechanism.

Nuclear Magnetic Resonance Spectroscopy Insights into Tau Structure in Solution: Impact of Post-translational Modifications.

Although Tau is an intrinsically disordered protein, some level of structure can still be defined, corresponding to short stretches of dynamic secondary structures and a preferential global fold described as an ensemble of conformations. These structures can be modified by Tau phosphorylation, and potentially other post-translational modifications. The analytical capacity of Nuclear Magnetic Resonance (NMR) spectroscopy provides the advantage of offering a residue-specific view of these modifications, allowing to link specific sites to a particular structure. The cis or trans conformation of X-Proline peptide bonds is an additional characteristic parameter of Tau structure that is targeted and modified by prolyl cis/trans isomerases. The challenge in molecular characterization of Tau lies in being able to link structural parameters to functional consequences in normal functions and dysfunctions of Tau, including potential misfolding on the path to aggregation and/or perturbation of the interactions of Tau with its many molecular partners. Phosphorylation of Ser and Thr residues has the potential to impact the local and global structure of Tau.

Global Folding of a Na+ -Specific DNAzyme Studied by FRET.

Recently, a few RNA-cleaving DNAzymes have been isolated with excellent specificity for Na+ , and some of them contain a Na+ -binding aptamer. This metal recognition mechanism is different from that of most previously reported DNAzymes. When using 2-aminopurine (2AP) as a probe, interesting local folding induced by Na+ was recently observed. In this work, FRET was used to probe the global folding of the Ce13d DNAzyme; one of the Na+ -specific DNAzymes. FRET pairs were at different locations, which yielded a total of five constructs to probe the three-way junction structure with a large loop. With endlabeled DNAzymes, the global structure appears to be quite rigid with little folding upon adding up to 200 mm monovalent metal ions, although some minor differences were observed between Li+ , Na+ , and K+ . This lack of significant conformational change is also consistent with circular dichroism spectroscopy data. The loop was then labeled with an internal tetramethylrhodamine fluorophore at the G14 position, and its cleavage activity was partially retained. A clear Na+ -dependent folding was observed with spectral crossover. From a biosensing standpoint, global folding based sensors are unlikely to work due to the overall rigid structure of the DNAzyme. Therefore, the best way to use this DNAzyme to discriminate Na+ from K+ is based on cleavage activity, followed by probing local folding, whereas global folding is the least effective for metal discrimination.

Sampling Native-like Structures of RNA-Protein Complexes through Rosetta Folding and Docking

RNA-protein complexes underlie numerous cellular processes including translation, splicing, and posttranscriptional regulation of gene expression. The structures of these complexes are crucial to their functions but often elude high-resolution structure determination. Computational methods are needed that can integrate low-resolution data for RNA-protein complexes while modeling de novo the large conformational changes of RNA components upon complex formation. To address this challenge, we describe RNP-denovo, a Rosetta method to simultaneously fold-and-dock RNA to a protein surface. On a benchmark set of diverse RNA-protein complexes not solvable with prior strategies, RNP-denovo consistently sampled native-like structures with better than nucleotide resolution. We revisited three past blind modeling challenges involving the spliceosome, telomerase, and a methyltransferase-ribosomal RNA complex in which previous methods gave poor results. When coupled with the same sparse FRET, crosslinking, and functional data used previously, RNP-denovo gave models with significantly improved accuracy. These results open a route to modeling global folds of RNA-protein complexes from low-resolution data.

Helix-Coil Transition Courses Through Multiple Pathways and Intermediates: Fast Kinetic Measurements and Dimensionality Reduction.

Nanosecond laser temperature jumps with tryptophan fluorescence detection and molecular dynamics simulation with kinetic dimensionality reduction were used to study the helix-coil transition in a 21-residue α-helical heteropeptide. Analysis of the temperature- dependent relaxation dynamics of this heteropeptide identified a distinct faster component of 20-35 ns, besides a slower component of 300-400 ns at temperatures between 296 and 280 K. To understand the mechanism of progression from a non-structured coil state to a structured helical state, we carried out a 12 μs molecular dynamics simulation of this peptide system. Clustering and optimal dimensionality reduction were applied to the molecular dynamics trajectory to generate low-dimensional coarse-grained models of the underlying kinetic network in terms of 2-5 metastable states. In accord with the generally accepted understanding of the multiple conformations and high entropy of the unfolded ensemble of states, the "coil" metastable set contains the largest number of structures. Interestingly, the helix metastable state was also found to be structurally heterogeneous, consisting of the completely helical form and several partly folded conformers that interconvert at a time scale faster that global folding. The intermediate states contain the fewest structures, have lowest populations, and have the shortest lifetimes. As the number of considered metastable states increases, more intermediates and more folding paths appear in the coarse-grained models. One of these intermediates corresponds to the transition state for folding, which involves an "off-center" helical region over residues 11-16. The kinetic network model is consistent with a statistical picture of folding following a simple reaction coordinate counting the helical population of individual residues. On the basis of simulations, we propose that the fast relaxation time should be assigned to cooperative folding/unfolding of segments of 1-4 neighboring residues.

The Cost of Long Catalytic Loops in Folding and Stability of the ALS-Associated Protein SOD1.

A conspicuous feature of the amyotrophic lateral sclerosis (ALS)-associated protein SOD1 is that its maturation into a functional enzyme relies on local folding of two disordered loops into a catalytic subdomain. To drive the disorder-to-order transition, the protein employs a single Zn2+ ion. The question is then if the entropic penalty of maintaining such disordered loops in the immature apoSOD1 monomer is large enough to explain its unusually low stability, slow folding, and pathological aggregation in ALS. To find out, we determined the effects of systematically altering the SOD1-loop lengths by protein redesign. The results show that the loops destabilize the apoSOD1 monomer by ∼3 kcal/mol, rendering the protein marginally stable and accounting for its aggregation behavior. Yet the effect on the global folding kinetics remains much smaller with a transition-state destabilization of <1 kcal/mol. Notably, this 1/3 transition-state to folded-state stability ratio provides a clear-cut example of the enigmatic disagreement between the Leffler α value from loop-length alterations (typically 1/3) and the "standard" reaction coordinates based on solvent perturbations (typically >2/3). Reconciling the issue, we demonstrate that the disagreement disappears when accounting for the progressive loop shortening that occurs along the folding pathway. The approach assumes a consistent Flory loop entropy scaling factor of c = 1.48 for both equilibrium and kinetic data and has the added benefit of verifying the tertiary interactions of the folding nucleus as determined by phi-value analysis. Thus, SOD1 not only represents a case where evolution of key catalytic function has come with the drawback of a destabilized apo state but also stands out as a well-suited model system for exploring the physicochemical details of protein self-organization.

Small Molecule Condensin Inhibitors.

Condensins play a unique role in orchestrating the global folding of the chromosome, an essential cellular process, and contribute to human disease and bacterial pathogenicity. As such, they represent an attractive and as yet untapped target for diverse therapeutic interventions. We describe here the discovery of small molecule inhibitors of the Escherichia coli condensin MukBEF. Pilot screening of a small diversity set revealed five compounds that inhibit the MukBEF pathway, two of which, Michellamine B and NSC260594, affected MukB directly. Computer-assisted docking suggested plausible binding sites for the two compounds in the hinge and head domains of MukB, and both binding sites were experimentally validated using mutational analysis and inspection of NSC260594 analogs. These results outline a strategy for the discovery of condensin inhibitors, identify druggable binding sites on the protein, and describe two small molecule inhibitors of condensins.

Multistep mutational transformation of a protein fold through structural intermediates.

New protein folds may evolve from existing folds through metamorphic evolution involving a dramatic switch in structure. To mimic pathways by which amino acid sequence changes could induce a change in fold, we designed two folded hybrids of Xfaso 1 and Pfl 6, a pair of homologous Cro protein sequences with ~40% identity but different folds (all-α vs. α + β, respectively). Each hybrid, XPH1 or XPH2, is 85% identical in sequence to its parent, Xfaso 1 or Pfl 6, respectively; 55% identical to its noncognate parent; and ~70% identical to the other hybrid. XPH1 and XPH2 also feature a designed hybrid chameleon sequence corresponding to the C-terminal region, which switched from α-helical to β-sheet structure during Cro evolution. We report solution nuclear magnetic resonance (NMR) structures of XPH1 and XPH2 at 0.3 Å and 0.5 Å backbone root mean square deviation (RMSD), respectively. XPH1 retains a global fold generally similar to Xfaso 1, and XPH2 retains a fold similar to Pfl 6, as measured by TM-align scores (~0.7), DALI Z-scores (7-9), and backbone RMSD (2-3 Å RMSD for the most ordered regions). However, these scores also indicate significant deviations in structure. Most notably, XPH1 and XPH2 have different, and intermediate, secondary structure content relative to Xfaso 1 and Pfl 6. The multistep progression in sequence, from Xfaso 1 to XPH1 to XPH2 to Pfl 6, thus involves both abrupt and gradual changes in folding pattern. The plasticity of some protein folds may allow for "polymetamorphic" evolution through intermediate structures.

Stabilizing Effect of Inherent Knots on Proteins Revealed by Molecular Dynamics Simulations

A growing number of proteins have been identified as knotted in their native structures, with such entangled topological features being expected to play stabilizing roles maintaining both the global fold and the nature of proteins. However, the molecular mechanism underlying the stabilizing effect is ambiguous. Here, we combine unbiased and mechanical atomistic molecular dynamics simulations to investigate how a protein is stabilized by an inherent knot by directly comparing chemical, thermal, and mechanical denaturing properties of two proteins having the same sequence and secondary structures but differing in the presence or absence of an inherent knot. One protein is YbeA from Escherichia coli, containing a deep trefoil knot within the sequence, and the other is the modified protein with the knot of YbeA being removed. Under certain chemical denaturing conditions, the unknotted protein fully unfolds whereas the knotted protein does not, suggesting a higher intrinsic stability for the protein having a knot. Both proteins unfold under enhanced thermal fluctuations but at different rates and with distinct pathways. Opening the hydrophobic core via separation between two α-helices is identified as a crucial step initiating the protein unfolding, which, however, is restrained for the knotted protein by topological and geometrical frustrations. Energy barriers for denaturing the protein are reduced by removing the knot, as evidenced by mechanical unfolding simulations. Finally, yet importantly, no obvious change in size or location of the knot was observed during denaturing processes, indicating that YbeA may remain knotted for a relatively long time during and after denaturation.