A Distinct Switch in Interactions of the Histone H4 Tail Domain upon Salt-dependent Folding of Nucleosome Arrays

The core histone tail domains mediate inter-nucleosomal interactions that direct folding and condensation of nucleosome arrays into higher-order chromatin structures. The histone H4 tail domain facilitates inter-array interactions by contacting both the H2A/H2B acidic patch and DNA of neighboring nucleosomes (1, 2). Likewise, H4 tail-H2A contacts stabilize array folding (3). However, whether the H4 tail domains stabilize array folding via inter-nucleosomal interactions with the DNA of neighboring nucleosomes remains unclear. We utilized defined oligonucleosome arrays containing a single specialized nucleosome with a photo-inducible cross-linker in the N terminus of the H4 tail to characterize these interactions. We observed that the H4 tail participates exclusively in intra-array interactions with DNA in unfolded arrays. These interactions are diminished during array folding, yet no inter-nucleosome, intra-array H4 tail-DNA contacts are observed in condensed chromatin. However, we document contacts between the N terminus of the H4 tail and H2A. Installation of acetylation mimics known to disrupt H4-H2A surface interactions did not increase observance of H4-DNA inter-nucleosomal interactions. These results suggest the multiple functions of the H4 tail require targeted distinct interactions within condensed chromatin.     

Role of Direct Interactions between the Histone H4 Tail and the H2A Core in Long Range Nucleosome Contacts

In eukaryotic nuclei the majority of genomic DNA is believed to exist in higher order chromatin structures. Nonetheless, the nature of direct, long range nucleosome interactions that contribute to these structures is poorly understood. To determine whether these interactions are directly mediated by contacts between the histone H4 amino-terminal tail and the acidic patch of the H2A/H2B interface, as previously demonstrated for short range nucleosomal interactions, we have characterized the extent and effect of disulfide cross-linking between residues in histones contained in different strands of nucleosomal arrays. We show that in 208-12 5 S rDNA and 601-177-12 nucleosomal array systems, direct interactions between histones H4-V21C and H2A-E64C can be captured. This interaction depends on the extent of initial cross-strand association but does not require these specific residues, because interactions with residues flanking H4-V21C can also be captured. Additionally, we find that trapping H2A-H4 intra-array interactions antagonizes the ability of these arrays to undergo intermolecular self-association.     

The 5.5 Protein of Phage T7 Inhibits H-NS through Interactions with the Central Oligomerization Domain

The 5.5 protein (T7p32) of coliphage T7 (5.5_T7) was shown to bind and inhibit gene silencing by the nucleoid-associated protein H-NS, but the mechanism by which it acts was not understood. The 5.5_T7 protein is insoluble when expressed in Escherichia coli, but we find that 5.5_T7 can be isolated in a soluble form when coexpressed with a truncated version of H-NS followed by subsequent disruption of the complex during anion-exchange chromatography. Association studies reveal that 5.5_T7 binds a region of H-NS (residues 60 to 80) recently found to contain a distinct domain necessary for higher-order H-NS oligomerization. Accordingly, we find that purified 5.5_T7 can disrupt higher-order H-NS-DNA complexes in vitro but does not abolish DNA binding by H-NS per se. Homologues of the 5.5_T7 protein are found exclusively among members of the Autographivirinae that infect enteric bacteria, and despite fairly low sequence conservation, the H-NS binding properties of these proteins are largely conserved. Unexpectedly, we find that the 5.5_T7 protein copurifies with heterogeneous low-molecular-weight RNA, likely tRNA, through several chromatography steps and that this interaction does not require the DNA binding domain of H-NS. The 5.5 proteins utilize a previously undescribed mechanism of H-NS antagonism that further highlights the critical importance that higher-order oligomerization plays in H-NS-mediated gene repression.     

Modulation of Folding Kinetics of Repeat Proteins: Interplay between Intra- and Interdomain Interactions

Repeat proteins have unique elongated structures that, unlike globular proteins, are quite modular. Despite their simple one-dimensional structure, repeat proteins exhibit intricate folding behavior with a complexity similar to that of globular proteins. Therefore, repeat proteins allow one to quantify fundamental aspects of the biophysics of protein folding. One important feature of repeat proteins is the interfaces between the repeating units. In particular, the distribution of stabilities within and between the repeats was previously suggested to affect their folding characteristics. In this study, we explore how the interface affects folding kinetics and cooperativity by investigating two families of repeat proteins, namely, the Ankyrin and tetratricopeptide repeat proteins, which differ in the number of interfacial contacts that are formed between their units as well as in their folding behavior. By using simple topology-based models, we show that modulating the energetic strength of the interface relative to that of the repeat itself can drastically change the protein stability, folding rate, and cooperativity. By further dissecting the interfacial contacts into several subsets, we isolated the effects of each of these groups on folding kinetics. Our study highlights the importance of interface connectivity in determining the folding behavior.     

Effects of Interactions with the GroEL Cavity on Protein Folding Rates

Encapsulation of proteins in chaperonins is an important mechanism by which the cell prevents the accumulation of misfolded species in the cytosol. However, results from theory and simulation for repulsive cavities appear to be inconsistent with recent experimental results showing, if anything, a slowdown in folding rate for encapsulated Rhodanese. We study the folding of Rhodanese in GroEL, using coarse-grained molecular simulations of the complete system including chaperonin and substrate protein. We find that, by approximating the substrate:GroEL interactions as repulsive, we obtain a strong acceleration in rate of between one and two orders of magnitude; a similar result is obtained by representing the chaperonin as a simple spherical cavity. Remarkably, however, we find that using a carefully parameterized, sequence-based potential to capture specific residue-residue interactions between Rhodanese and the GroEL cavity walls induces a very strong reduction of the folding rates. The effect of the interactions is large enough to completely offset the effects of confinement, such that folding in some cases can be even slower than that of the unconfined protein. The origin of the slowdown appears to be stabilization—relative to repulsive confinement—of the unfolded state through binding to the cavity walls, rather than a reduction of the diffusion coefficient along the folding coordinate.     

Cotranslational Translocation and Folding of a Periplasmic Protein Domain in Escherichia coli

In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) – a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide – to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB’s two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45–50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm.     

Biophysical Analysis of the MHR Motif in Folding and Domain Swapping of the HIV Capsid Protein C-Terminal Domain

Infection by human immunodeficiency virus (HIV) depends on the function, in virion morphogenesis and other stages of the viral cycle, of a highly conserved structural element, the major homology region (MHR), within the carboxyterminal domain (CTD) of the capsid protein. In a modified CTD dimer, MHR is swapped between monomers. While no evidence for MHR swapping has been provided by structural models of retroviral capsids, it is unknown whether it may occur transiently along the virus assembly pathway. Whatever the case, the MHR-swapped dimer does provide a novel target for the development of anti-HIV drugs based on the concept of trapping a nonnative capsid protein conformation. We have carried out a thermodynamic and kinetic characterization of the domain-swapped CTD dimer in solution. The analysis includes a dissection of the role of conserved MHR residues and other amino acids at the dimerization interface in CTD folding, stability, and dimerization by domain swapping. The results revealed some energetic hotspots at the domain-swapped interface. In addition, many MHR residues that are not in the protein hydrophobic core were nevertheless found to be critical for folding and stability of the CTD monomer, which may dramatically slow down the swapping reaction. Conservation of MHR residues in retroviruses did not correlate with their contribution to domain swapping, but it did correlate with their importance for stable CTD folding. Because folding is required for capsid protein function, this remarkable MHR-mediated conformational stabilization of CTD may help to explain the functional roles of MHR not only during immature capsid assembly but in other processes associated with retrovirus infection. This energetic dissection of the dimerization interface in MHR-swapped CTD may also facilitate the design of anti-HIV compounds that inhibit capsid assembly by conformational trapping of swapped CTD dimers.     

Consequences of Domain Insertion on the Stability and Folding Mechanism of a Protein

SlyD, the sensitive-to-lysis protein from Escherichia coli, consists of two domains. They are not arranged successively along the protein chain, but one domain, the “insert-in-flap” (IF) domain, is inserted internally as a guest into a surface loop of the host domain, which is a prolyl isomerase of the FK506 binding protein (FKBP) type. We used SlyD as a model to elucidate how such a domain insertion affects the stability and folding mechanism of the host and the guest domain. For these studies, the two-domain protein was compared with a single-domain variant SlyDΔIF, SlyD* without the chaperone domain (residues 1-69 and 130-165) in which the IF domain was removed and replaced by a short loop, as present in human FKBP12. Equilibrium unfolding and folding kinetics followed an apparent two-state mechanism in the absence and in the presence of the IF domain. The inserted domain decreased, however, the stability of the host domain in the transition region and decelerated its refolding reaction by about 10-fold. This originates from the interruption of the chain connectivity by the IF domain and its inherent instability. To monitor folding processes in this domain selectively, a Trp residue was introduced as fluorescent probe. Kinetic double-mixing experiments revealed that, in intact SlyD, the IF domain folds and unfolds about 1000-fold more rapidly than the FKBP domain, and that it is strongly stabilized when linked with the folded FKBP domain. The unfolding limbs of the kinetic chevrons of SlyD show a strong downward curvature. This deviation from linearity is not caused by a transition-state movement, as often assumed, but by the accumulation of a silent unfolding intermediate at high denaturant concentrations. In this kinetic intermediate, the FKBP domain is still folded, whereas the IF domain is already unfolded.     

The Measles Virus Nucleocapsid Protein Tail Domain Is Dispensable for Viral Polymerase Recruitment and Activity

Paramyxovirus genomes are ribonucleoprotein (RNP) complexes consisting of nucleoprotein (N)-encapsidated viral RNA. Measles virus (MeV) N features an amino-terminal RNA-binding core and a 125-residue tail domain, of which only the last 75 residues are considered fully mobile on the nucleocapsid surface. A molecular recognition element (MoRE) domain mediates binding of the viral phosphoprotein (P). This P N-tail interaction is considered instrumental for recruiting the polymerase complex to the template. We have engineered MeV N variants with tail truncations progressively eliminating the MoRE domain and upstream tail sections. Confirming previous reports, RNPs with N truncations lacking the carboxyl-terminal 43-residues harboring the MoRE domain cannot serve as polymerase template. Remarkably, further removal of all tail residues predicted to be surface-exposed significantly restores RNP bioactivity. Insertion of structurally dominant tags into the central N-tail section reduces bioactivity, but the negative regulatory effect of exposed N-tail stems is sequence-independent. Bioactive nucleocapsids lacking exposed N-tail sections are unable to sustain virus replication, because of weakened interaction of the advancing polymerase complex with the template. Deletion of the N-MoRE-binding domain in P abrogates polymerase recruitment to standard nucleocapsids, but polymerase activity is partially restored when N-tail truncated RNPs serve as template. Revising central elements of the current replication model, these data reveal that MeV polymerase is capable of productively docking directly to the nucleocapsid core. Dispensable for polymerase recruitment, N-MoRE binding to P-tail stabilizes the advancing polymerase-RNP complex and may rearrange unstructured central tail sections to facilitate polymerase access to the template.     

Nonnative Electrostatic Interactions Can Modulate Protein Folding: Molecular Dynamics with a Grain of Salt

In recent years, a growing number of protein folding studies have focused on the unfolded state, which is now recognized as playing a major role in the folding process. Some of these studies show that interactions occurring in the unfolded state can significantly affect the stability and kinetics of the protein folding reaction. In this study, we modeled the effect of electrostatic interactions, both native and nonnative, on the folding of three protein systems that underwent selective charge neutralization or reversal or complete charge suppression. In the case of the N-terminal L9 protein domain, our results directly attribute the increase in thermodynamic stability to destabilization of the unfolded ensemble, reaffirming the experimental observations. These results provide a deeper structural insight into the ensemble of the unfolded state and predict a new mutation site for increased protein stability. In the second case, charge reversal mutations of RNase Sa affected protein stability, with the destabilizing mutations being less destabilizing at higher salt concentrations, indicating the formation of charge-charge interactions in the unfolded state. In the N-terminal L9 and RNase Sa systems, changes in electrostatic interactions in the unfolded state that cause an increase in free energy had an overall compaction effect that suggests a decrease in entropy. In the third case, in which we compared the β-lactalbumin and hen egg-white lysozyme protein homologues, we successfully eliminated differences between the folding kinetics of the two systems by suppressing electrostatic interactions, supporting previously reported findings. Our coarse-grained molecular dynamics study not only reproduces experimentally reported findings but also provides a detailed molecular understanding of the elusive unfolded-state ensemble and how charge-charge interactions can modulate the biophysical characteristics of folding.     

The Role of Backbone Hydrogen Bonds in the Transition State for Protein Folding of a PDZ Domain

Backbone hydrogen bonds are important for the structure and stability of proteins. However, since conventional site-directed mutagenesis cannot be applied to perturb the backbone, the contribution of these hydrogen bonds in protein folding and stability has been assessed only for a very limited set of small proteins. We have here investigated effects of five amide-to-ester mutations in the backbone of a PDZ domain, a 90-residue globular protein domain, to probe the influence of hydrogen bonds in a β-sheet for folding and stability. The amide-to-ester mutation removes NH-mediated hydrogen bonds and destabilizes hydrogen bonds formed by the carbonyl oxygen. The overall stability of the PDZ domain generally decreased for all amide-to-ester mutants due to an increase in the unfolding rate constant. For this particular region of the PDZ domain, it is therefore clear that native hydrogen bonds are formed after crossing of the rate-limiting barrier for folding. Moreover, three of the five amide-to-ester mutants displayed an increase in the folding rate constant suggesting that the hydrogen bonds are involved in non-native interactions in the transition state for folding.     

Structural Characterizations of the Fas Receptor and the Fas-Associated Protein with Death Domain Interactions

The Fas receptor is a representative death receptor, and the Fas-associated protein with death domain (FADD) is a crucial adapter protein needed to support the Fas receptor’s activity. The Fas–FADD interactions constitute an important signaling pathway that ultimately induces apoptosis or programmed cell death in biological systems. The interactions responsible for this cell-death process are governed by the binding process of the Fas ligand to the Fas, followed by the caspase cascade activation. Using a computational approach, the present communication explores certain essential structural aspects of the Fas–FADD death domains and their interfacial interactions.     

Solution Scattering and FRET Studies on Nucleosomes Reveal DNA Unwrapping Effects of H3 and H4 Tail Removal

Using a combination of small-angle X-ray scattering (SAXS) and fluorescence resonance energy transfer (FRET) measurements we have determined the role of the H3 and H4 histone tails, independently, in stabilizing the nucleosome DNA terminal ends from unwrapping from the nucleosome core. We have performed solution scattering experiments on recombinant wild-type, H3 and H4 tail-removed mutants and fit all scattering data with predictions from PDB models and compared these experiments to complementary DNA-end FRET experiments. Based on these combined SAXS and FRET studies, we find that while all nucleosomes exhibited DNA unwrapping, the extent of this unwrapping is increased for nucleosomes with the H3 tails removed but, surprisingly, decreased in nucleosomes with the H4 tails removed. Studies of salt concentration effects show a minimum amount of DNA unwrapping for all complexes around 50-100mM of monovalent ions. These data exhibit opposite roles for the positively-charged nucleosome tails, with the ability to decrease access (in the case of the H3 histone) or increase access (in the case of the H4 histone) to the DNA surrounding the nucleosome. In the range of salt concentrations studied (0-200mM KCl), the data point to the H4 tail-removed mutant at physiological (50-100mM) monovalent salt concentration as the mononucleosome with the least amount of DNA unwrapping.