The FACT Spt16 "peptidase" domain is a histone H3-H4 binding module

The FACT complex is a conserved cofactor for RNA polymerase II elongation through nucleosomes. FACT bears histone chaperone activity and contributes to chromatin integrity. However, the molecular mechanisms behind FACT function remain elusive. Here we report biochemical, structural, and mutational analyses that identify the peptidase homology domain of the Schizosaccharomyces pombe FACT large subunit Spt16 (Spt16-N) as a binding module for histones H3 and H4. The 2.1-A crystal structure of Spt16-N reveals an aminopeptidase P fold whose enzymatic activity has been lost. Instead, the highly conserved fold directly binds histones H3-H4 through a tight interaction with their globular core domains, as well as with their N-terminal tails. Mutations within a conserved surface pocket in Spt16-N or posttranslational modification of the histone H4 tail reduce interaction in vitro, whereas the globular domains of H3-H4 and the H3 tail bind distinct Spt16-N surfaces. Our analysis suggests that the N-terminal domain of Spt16 may add to the known H2A-H2B chaperone activity of FACT by including a H3-H4 tail and H3-H4 core binding function mediated by the N terminus of Spt16. We suggest that these interactions may aid FACT-mediated nucleosome reorganization events.     

Histone H4 acetylation by immunohistochemistry and prognosis in relapsed acute lymphocytic leukaemia (ALL)

Histone H4 acetylation was examined by immunohistochemistry in patients with acute lymphocytic leukaemia (ALL) in first relapse. Univariate and multivariate models identified correlates of complete remission (CR) and overall survival (OS). No variables were associated with achievement of CR. In multivariate analysis, weak histone H4 acetylation [Hazard Ratio (HR) 2·20, 95% confidence interval (CI) 0·93–5·23, P = 0·07], shorter interval from diagnosis to relapse (<9 vs. 9–24 vs. >24 months) (HR 1·82, 95% CI 1·20–2·75, P = 0·005), and central nervous system involvement (HR 3·43, 95% CI 1·31–8·99, P = 0·01) were independent poor prognostic factors for OS. These data provide a rationale for the use of histone deacetylase inhibitors in the treatment of relapsed ALL.     

Energy landscape views for interplays among folding,binding, and allostery of calmodulin domains

Ligand binding modulates the energy landscape of proteins, thus altering their folding and allosteric conformational dynamics. To investigate such interplay, calmodulin has been a model protein. Despite much attention, fully resolved mechanisms of calmodulin folding/binding have not been elucidated. Here, by constructing a computational model that can integrate folding, binding, and allosteric motions, we studied in-depth folding of isolated calmodulin domains coupled with binding of two calcium ions and associated allosteric conformational changes. First, mechanically pulled simulations revealed coexistence of three distinct conformational states: the unfolded, the closed, and the open states, which is in accord with and augments structural understanding of recent single-molecule experiments. Second, near the denaturation temperature, we found the same three conformational states as well as three distinct binding states: zero, one, and two calcium ion bound states, leading to as many as nine states. Third, in terms of the nine-state representation, we found multiroute folding/binding pathways and shifts in their probabilities with the calcium concentration. At a lower calcium concentration, “combined spontaneous folding and induced fit” occurs, whereas at a higher concentration, “binding-induced folding” dominates. Even without calcium binding, we observed that the folding pathway of calmodulin domains can be modulated by the presence of metastable states. Finally, full-length calmodulin also exhibited an intriguing coupling between two domains when applying tension.Protein folding and conformational dynamics have often been characterized by the energy landscape of proteins (1–5). The energy landscape is dependent on the molecular physiochemistry and thus is modulated by many factors, such as chemical modification and ligand binding. Ligand binding, in turn, is dependent on the conformation of proteins. Thus, folding, binding, and allosteric conformational dynamics are mutually correlated. Despite their obvious correlation in concept, it has been very challenging to characterize how they are indeed coupled for any single proteins. Here, we address, in depth, how these three types of dynamics, folding, binding, and allosteric conformational dynamics, are coupled from the energy landscape perspective for a specific protein, calmodulin (CaM).CaM is a ubiquitous calcium-binding messenger protein involved in signal transduction (6) and, more importantly here, has been a model protein to investigate folding, binding, and allostery. Full-length CaM has two nearly symmetric globular domains connected by a flexible central helix (7, 8). Each domain is composed of paired EF hands containing two Ca²⁺-binding sites (Fig. 1A). Upon binding to Ca²⁺, each CaM domain undergoes substantial conformational change from a closed state to an open state, exposing a hydrophobic patch that can bind with target proteins and regulate downstream processes (9). CaM has been frequently used as a model in studying the folding of multidomain proteins (10, 11), allosteric transitions (12–14), slow conformational dynamics around physiological temperatures (15–18), metal ion binding (19, 20), and correlation between inherent flexibility and protein functions (21, 22). For example, using structure-based coarse-grained (CG) simulations, Chen and Hummer elucidated the coexistence of an unfolded state, a closed state, and an open state around physiological temperatures for the C-terminal domain of CaM (CaM-C) without Ca²⁺ binding (15), which reconciles some seemingly contradictory experimental observations on the slow conformational dynamics of CaM.Open in a separate windowFig. 1.(A) Three-dimensional structure of calmodulin domain at closed [Protein Data Bank (PDB) code: 1cfd] and open states (PDB code:1cll). Calcium ions are represented by yellow spheres. (B) Schematic of coupling among folding, calcium binding, and allosteric motions for the CaM domain. Due to the conformational transitions between open and closed states, in addition to the direct folding pathway (red solid arrow), folding to the most stable state may involve an alternative pathway via a metastable state (green arrow plus blue arrow). The calcium binding can modulate the relative stability of the conformational states and therefore the population of folding pathways. O, C, and U represent open, closed, and unfolded states, respectively.More recently, Rief and coworkers studied the Ca²⁺-dependent folding of CaM based on a new generation technique of single-molecule force spectroscopy, which can probe the reversible folding/unfolding transitions with near equilibrium conditions (10, 23, 24). Their results revealed that at high Ca²⁺ concentrations, the folding pathway of the CaM domain proceeds via a transition state capable of binding Ca²⁺ ions, demonstrating the coupling between Ca²⁺ binding and CaM folding. All these computational and experimental works provided unprecedented understanding of many aspects of the folding and allosteric transitions of CaM. However, a full picture of the coupling among folding, Ca²⁺ binding, and allosteric motions, as schematically shown in Fig. 1B, is still lacking. Particularly, two fundamental issues arising from the allostery and Ca²⁺-binding characteristics of CaM remain elusive: (i) How does the allosteric feature of the energy landscape contribute to the folding complexity? And (ii) how can the folding mechanism of CaM be modulated by Ca²⁺ binding?Motivated by previous computational and experimental studies (15, 23), in this work we investigated the folding coupled with Ca²⁺ binding and allosteric motions of the isolated CaM domains as well as the full-length CaM. To do so, we first integrated computational tools developed for folding, ligand binding, and allosteric motions together. The proposed CG protein model was used for the subsequent series of molecular dynamics (MD) simulations. First, corresponding to Rief’s experiments, we performed MD simulations of isolated CaM domains with pretensions, which gave consistent results with the experiments and, in addition, provided the direct structural assignment on the experimentally observed states. Second, at a higher temperature, without pretension we performed reversible folding/unfolding simulations for a wide range of Ca²⁺ concentrations. The conformational and ligand-binding energy landscape revealed as many as nine distinctive states. Then, we analyzed the binding-coupled folding reactions in terms of the nine states, finding multiple routes and their modulation by Ca²⁺ concentrations. Interestingly, as the Ca²⁺ concentration increases, the CaM domain folding mechanism switches from “combined spontaneous folding and induced fit” to “binding-induced folding,” which accords with the scenario deduced from single-molecule force spectroscopy experiments. Finally, the effects of tension on the conformational fluctuations of the full-length CaM are discussed.     

Heterochromatin protein Sir3 induces contacts between the amino terminus of histone H4 and nucleosomal DNA

The regulated binding of effector proteins to the nucleosome plays a central role in the activation and silencing of eukaryotic genes. How this binding changes the properties of chromatin to mediate gene activation or silencing is not fully understood. Here we provide evidence that association of the budding yeast silent information regulator 3 (Sir3) silencing protein with the nucleosome induces a conformational change in the amino terminus of histone H4 that promotes interactions between the conserved H4 arginines 17 and 19 (R17 and R19) and nucleosomal DNA. Substitutions of H4R17 and R19 with alanine abolish silencing in vivo, but have little or no effect on binding of Sir3 to nucleosomes or histone H4 peptides in vitro. Furthermore, in both the previously reported crystal structure of the Sir3-bromo adjacent homology (BAH) domain bound to the Xenopus laevis nucleosome core particle and the crystal structure of the Sir3-BAH domain bound to the yeast nucleosome core particle described here, H4R17 and R19 make contacts with nucleosomal DNA rather than with Sir3. These results suggest that Sir3 binding generates a more stable nucleosome by clamping H4R17 and R19 to nucleosomal DNA, and raise the possibility that such induced changes in histone–DNA contacts play major roles in the regulation of chromatin structure.     

Topography of funneled landscapes determines the thermodynamics and kinetics of protein folding

The energy landscape approach has played a fundamental role in advancing our understanding of protein folding. Here, we quantify protein folding energy landscapes by exploring the underlying density of states. We identify three quantities essential for characterizing landscape topography: the stabilizing energy gap between the native and nonnative ensembles δE, the energetic roughness ΔE, and the scale of landscape measured by the entropy S. We show that the dimensionless ratio between the gap, roughness, and entropy of the system accurately predicts the thermodynamics, as well as the kinetics of folding. Large Λ implies that the energy gap (or landscape slope towards the native state) is dominant, leading to more funneled landscapes. We investigate the role of topological and energetic roughness for proteins of different sizes and for proteins of the same size, but with different structural topologies. The landscape topography ratio Λ is shown to be monotonically correlated with the thermodynamic stability against trapping, as characterized by the ratio of folding temperature versus trapping temperature. Furthermore, Λ also monotonically correlates with the folding kinetic rates. These results provide the quantitative bridge between the landscape topography and experimental folding measurements.     

Proton-coupled electron transfer and the role of water molecules in proton pumping by cytochrome c oxidase

Molecular oxygen acts as the terminal electron sink in the respiratory chains of aerobic organisms. Cytochrome c oxidase in the inner membrane of mitochondria and the plasma membrane of bacteria catalyzes the reduction of oxygen to water, and couples the free energy of the reaction to proton pumping across the membrane. The proton-pumping activity contributes to the proton electrochemical gradient, which drives the synthesis of ATP. Based on kinetic experiments on the O–O bond splitting transition of the catalytic cycle (A → P_R), it has been proposed that the electron transfer to the binuclear iron–copper center of O₂ reduction initiates the proton pump mechanism. This key electron transfer event is coupled to an internal proton transfer from a conserved glutamic acid to the proton-loading site of the pump. However, the proton may instead be transferred to the binuclear center to complete the oxygen reduction chemistry, which would constitute a short-circuit. Based on atomistic molecular dynamics simulations of cytochrome c oxidase in an explicit membrane–solvent environment, complemented by related free-energy calculations, we propose that this short-circuit is effectively prevented by a redox-state–dependent organization of water molecules within the protein structure that gates the proton transfer pathway.Life on Earth is supported by a constant supply of energy in the form of ATP. Cytochrome c oxidase (CcO) in the respiratory chains of mitochondria and bacteria catalyzes the exergonic reduction of molecular oxygen (O₂) to water and uses the free energy of the reaction to pump protons across the membrane (1–3). The oxygen reduction reaction takes place at a highly conserved active site formed by two metal sites, heme a₃ and Cu_B (Fig. 1 A and B), called the binuclear center (BNC). The electrons donated by the mobile electron carrier cytochrome c reach the BNC via two other conserved metal centers, Cu_A and heme a (Fig. 1A). The protons required for the chemistry of O₂ reduction to water, and for proton pumping, are transported with the assistance of side chains of polar amino acids and conserved water molecules in the protein interior (4–6) (Fig. 1A). Two such proton transfer pathways have been described in the mitochondrial and bacterial A-type oxidases (to distinguish between different types of oxidases, see ref. 7), namely, the D and K channels (8, 9), the names of which are based on the conserved amino acid residues Asp91 and Lys319, respectively (Fig. 1A, amino acid numbering based on the bovine heart CcO). The D channel is responsible for the translocation of all of the pumped protons, and for the transfer of at least two of the four protons required for oxygen reduction chemistry, whereas the K channel supplies one or two protons to the BNC during the reductive phase of the catalytic cycle (8, 9). The D channel terminates at a highly conserved glutamic acid residue, Glu242, from where the protons are either transferred to the BNC for consumption, or to the proton-loading site (PLS) for pumping across the membrane (Fig. 1A). In 2003, Wikström et al. postulated a molecular mechanism in which water molecules in the nonpolar cavity above Glu242 would form proton-transferring chains, the orientation of which depends upon the redox state of the enzyme (10). They proposed that the reduction of the low-spin heme would result in transfer of a proton via a preorganized water chain from Glu242 to the d-propionate (Dprp) of the high-spin heme, whereas in the case when the electron has moved to the BNC, the water chain would reorientate and conduct protons from Glu242 to the BNC (Fig. 1A, and see below). Even though there is little direct experimental support available for such a water-gated mechanism, a recent FTIR study indeed suggests changes in water organization upon changes in the redox state of the enzyme (11). Many of the elementary steps that were postulated in the water-gated mechanism have gained support from experiments in the recent past (12, 13).Open in a separate windowFig. 1.(A) A three-subunit (SU) CcO. SU I (blue), II (red), and III (orange) are displayed as transparent ribbons. The D and K channels of proton transfer are marked with blue arrows. Crystallographic water molecules present in these proton channels are shown in purple. Electron transfer (red arrow) takes place from Cu_A (orange) via heme a (yellow) to the binuclear center comprising heme a₃ (yellow)–Cu_B (orange). Protons are transferred from Glu242 (E242) either to the PLS or to the binuclear center (black arrows). Lipid bilayer (silver lines), water (gray dots), and sodium (light yellow) and chloride (cyan) ions are also displayed. (B) The catalytic cycle of CcO. The states of heme a₃, Cu_B, and the cross-linked tyrosine are displayed. Each light orange rectangle corresponds to a state of the BNC, the name of which is displayed in red (Upper Right). Pumped protons are shown in blue, black H⁺ indicates uptake of a proton for water formation, and e⁻ indicates transfer of an electron from the low-spin heme a. Catalysis of O₂ reduction occurs clockwise.It is generally thought that the proton pump of CcO operates via the same mechanism in each of the 4 one-electron reduction steps of the catalytic cycle (Fig. 1B). However, kinetic data on two different transitions (A → P_R and O_H → E_H) have suggested dissimilarities in some of the elementary steps (12, 13). Fully reduced enzyme reacts with oxygen and forms an oxygenated adduct A in ca. 10 µs, followed by splitting of the O–O bond leading to formation of the P_R intermediate (in ∼25 μs) that is linked to loading of the PLS with a proton (3, 12). O–O bond splitting from state A in the absence of electrons in heme a or Cu_A yields the stable state P_M without proton transfer to the PLS (3, 12). Therefore, it is the electron transfer from heme a into the BNC accompanying O–O bond scission during A → P_R that is linked to the proton transfer to the PLS. The structure of the P_R intermediate is well characterized with ferryl heme a₃, cupric hydroxide, and tyrosinate (3, 14). In P_M the tyrosine is almost certainly in the form of a neutral radical (3, 14), so the reaction P_M → P_R is a proton-coupled electron transfer reaction (PCET) that initiates the reactions of the proton pump (3, 12). Note that in the state P_R the proton at the PLS partially neutralizes the electron in the BNC (3) in accordance with the charge-neutralization principle of the BNC (15). However, an important question arises: how can proton transfer from Glu242 to the BNC be prevented, which would short-circuit one step of proton pumping and form the next stable intermediate F? In the O_H → E_H transition of the catalytic cycle this short-circuit is minimized because reduction of the low-spin heme is thought to raise the pK_a of the PLS sufficiently to lead to its protonation before transfer of the electron to the BNC (3, 10, 13, 16–18), and uncompensated proton transfer to the BNC is endergonic in nature (refs. 13,16,17; cf. ref. 19). In contrast, the likelihood of a proton leak in the A → P_R transition increases manifold because the electron transfer from heme a to the BNC is required for loading of the PLS with a proton (3, 12). This facet is analyzed in the current work, and it is proposed that it is the orientation of the water molecules in the nonpolar cavity above Glu242 that effectively gates the pump and minimizes such a short-circuit.     

Coevolutionary information,protein folding landscapes,and the thermodynamics of natural selection

The energy landscape used by nature over evolutionary timescales to select protein sequences is essentially the same as the one that folds these sequences into functioning proteins, sometimes in microseconds. We show that genomic data, physical coarse-grained free energy functions, and family-specific information theoretic models can be combined to give consistent estimates of energy landscape characteristics of natural proteins. One such characteristic is the effective temperature T_sel at which these foldable sequences have been selected in sequence space by evolution. T_sel quantifies the importance of folded-state energetics and structural specificity for molecular evolution. Across all protein families studied, our estimates for T_sel are well below the experimental folding temperatures, indicating that the energy landscapes of natural foldable proteins are strongly funneled toward the native state.The physics and natural history of proteins are inextricably intertwined (1, 2). The cooperative manner in which proteins find their way to a folded structure is the result of proteins having undergone natural selection and not typical of random polymers (3, 4). Likewise, the requirement that most proteins must fold to function is a strong constraint on their phylogeny. The unavoidable random mutation events that proteins have undergone throughout their evolution have provided countless numbers of physicochemical experiments on folding landscapes. Thus, the evolutionary patterns of proteins found through comparative sequence analysis can be used to understand protein structure and energetics. In this paper, we compare the information content in the correlated changes that have occurred in protein sequences of common ancestry with energies from a transferable energy function to quantify the influence of maintaining foldability on molecular evolution.     

H3K4 methyltransferase Set1 is involved in maintenance of ergosterol homeostasis and resistance to Brefeldin A

Set1 is a conserved histone H3 lysine 4 (H3K4) methyltransferase that exists as a multisubunit complex. Although H3K4 methylation is located on many actively transcribed genes, few studies have established a direct connection showing that loss of Set1 and H3K4 methylation results in a phenotype caused by disruption of gene expression. In this study, we determined that cells lacking Set1 or Set1 complex members that disrupt H3K4 methylation have a growth defect when grown in the presence of the antifungal drug Brefeldin A (BFA), indicating that H3K4 methylation is needed for BFA resistance. To determine the role of Set1 in BFA resistance, we discovered that Set1 is important for the expression of genes in the ergosterol biosynthetic pathway, including the rate-limiting enzyme HMG-CoA reductase. Consequently, deletion of SET1 leads to a reduction in HMG-CoA reductase protein and total cellular ergosterol. In addition, the lack of Set1 results in an increase in the expression of DAN1 and PDR11, two genes involved in ergosterol uptake. The increase in expression of uptake genes in set1Δ cells allows sterols such as cholesterol and ergosterol to be actively taken up under aerobic conditions. Interestingly, when grown in the presence of ergosterol set1Δ cells become resistant to BFA, indicating that proper ergosterol levels are needed for antifungal drug resistance. These data show that H3K4 methylation impacts gene expression and output of a biologically and medically relevant pathway and determines why cells lacking H3K4 methylation have antifungal drug sensitivity.     

Complexity of chromatin folding is captured by the strings and binders switch model

Chromatin has a complex spatial organization in the cell nucleus that serves vital functional purposes. A variety of chromatin folding conformations has been detected by single-cell imaging and chromosome conformation capture-based approaches. However, a unified quantitative framework describing spatial chromatin organization is still lacking. Here, we explore the “strings and binders switch” model to explain the origin and variety of chromatin behaviors that coexist and dynamically change within living cells. This simple polymer model recapitulates the scaling properties of chromatin folding reported experimentally in different cellular systems, the fractal state of chromatin, the processes of domain formation, and looping out. Additionally, the strings and binders switch model reproduces the recently proposed “fractal–globule” model, but only as one of many possible transient conformations.     

Physicochemical properties of the cytoplasmic triiodothyronine binding protein from tadpole liver and tail fin

The sedimentation velocity, gel filtration properties and pattern of elution from ion exchange gels of the cytoplasmic triiodothyronine (T₃) binding protein from Rana catesbeiana liver and tail fin cytosol were determined. The T₃ binding protein in liver cytosol had a sedimentation coefficient of 4.4S on sucrose gradients and a Stokes radius from Sephadex gel filtration of 38.2 ± 4.2 A. From these two values a molecular weight of 71,700 ± 4100 and a frictional ratio of 1.28 ± 0.05 for the T₃ binding protein from tadpole liver has been calculated. The liver T₃ binding protein was eluted from DEAE-Sephadex gels at a salt concentration of 0.233 ± 0.017 M NaCl. The sedimentation coefficient, Stokes radius and salt requirement for elution from DEAE-Sephadex of the T₃ binding protein from tail fin cytosol were essentially identical (4.4S, $\text{35.2 ± 4.2}\text{A}\text{?}\text{and}\text{0.213 ± 0.015}\text{M NaCl}$ respectively). The calculated molecular weight is 66,100 ± 7900 and the frictional ratio is 1.21 ± 0.10 for the tail fin T₃ binding protein. The great similarity in the physicochemical properties of the T₃ binding protein from the liver and tail fin implies that the T₃ binding protein in each tissue is similar if not identical. The possible reason for the differences in the dissociation constants previously reported for the binding of T₃ in the two tissues is discussed.