Essential domain motions in barnase revealed by MD simulations

The wealth of data accumulated on the bacterial ribonuclease barnase is complemented by molecular dynamics trajectories starting from four different experimental structures and covering a total of >10 ns. Using principal component analysis, the simulations are interpreted in view of dynamic domains and hinges promoting relative motions of these domains. Two domains with residues 7-22 and 52-108 for the first domain and residues 25-51 for the second domain were consistently observed. Hinge regions consist primarily of Tyr24, Ser50, Ile51, and Gly52. Earlier mutation studies have demonstrated that the residues of the hinge regions play essential roles for the stability and activity of barnase. The domain motions are correlated to inter-domain interactions involving functionally important active site residues, such as Lys27 and Glu73. A model is presented that combines the observation of dynamic domains and their motions with the extensive mutation data from the literature. Enthalpic energy contributions originating from specific inter-domain interactions as well as entropic energy contributions due to the domain motions are discussed in the frame of this model and compared with destabilization energies measured for corresponding mutants.     

p53 chromatin epigenetic domain organization and p53 transcription

Epigenetic organization represents an important regulation mechanism of gene expression. In this work, we show that the mouse p53 gene is organized into two epigenetic domains. The first domain is fully unmethylated, associated with histone modifications in active genes, and organized in a nucleosome-free conformation that is deficient in H2a/H2b, whereas the second domain is fully methylated, associated with deacetylated histones, and organized in a nucleosomal structure. In mitotic cells, RNA polymerase is depleted in domain II, which is folded into a higher-order structure and is associated with H1 histone, whereas domain I conformation is preserved. Similar results were obtained for cells treated with inhibitors of associated regulatory factors. These results suggest that depletion of RNA polymerase II is the result of a physical barrier due to the folding of chromatin in domain II. The novel chromatin structure in the first domain during mitosis also suggests a mechanism for marking active genes in successive cell cycles.     

Structural stability and domain organization of colicin E1

Thermodynamic properties, stability, and structure of the toxin-like molecule colicin E1 were analyzed by differential scanning calorimetry and circular dichroism to determine the number of structurally independent domains, and the interdomain interactions necessary for colicin import into the Escherichia coli cell. Analysis of denaturation profiles of the 522 residue colicin E1, together with fragments of 342 and 178 residues that contain subsets of the domains, showed three stable cooperative blocks that differ in thermal stability and correspond to three major functional domains of the colicin: (i) the COOH-terminal channel-forming (C) domain with the highest thermal stability; (ii) the BtuB receptor binding (R) domain; and (iii) the N-terminal translocation (T) domain that has the smallest stabilization enthalpy and thermal stability. Interdomain interactions were described in which T-R interactions stabilize R, and T-C and R-C interactions stabilize R and T, but destabilize C. The R and T domains behaved in a similar way as a function of pH and ionic strength. Interacting extended helices of the R domain, possibly a coiled-coil, were implied by: (i) the very high (>90%) alpha-helical content of the R domain, (ii) cooperative decreases in alpha-helical content near the T(tr) of thermal denaturation of the R domain; (iii) a large denaturation enthalpy, implying extensive H-bond and van der Waals interactions. The R domain was inferred, from the extended network of interacting helices, large DeltaH, and steep temperature dependence of its stabilization energy to have a dominant role in determining the conformation of other domains. It is proposed that cellular import starts with the R domain binding to the BtuB receptor, followed by unfolding of the R domain coiled-coil and thereby of the T domain, which then interacts with the TolC receptor-translocator.     

Membrane-binding mechanism of the EEA1 FYVE domain revealed by multi-scale molecular dynamics simulations

Early Endosomal Antigen 1 (EEA1) is a key protein in endosomal trafficking and is implicated in both autoimmune and neurological diseases. The C-terminal FYVE domain of EEA1 binds endosomal membranes, which contain phosphatidylinositol-3-phosphate (PI(3)P). Although it is known that FYVE binds PI(3)P specifically, it has not previously been described of how FYVE attaches and binds to endosomal membranes. In this study, we employed both coarse-grained (CG) and atomistic (AT) molecular dynamics (MD) simulations to determine how FYVE binds to PI(3)P-containing membranes. CG-MD showed that the dominant membrane binding mode resembles the crystal structure of EEA1 FYVE domain in complex with inositol-1,3-diphospate (PDB ID 1JOC). FYVE, which is a homodimer, binds the membrane via a hinge mechanism, where the C-terminus of one monomer first attaches to the membrane, followed by the C-terminus of the other monomer. The estimated total binding energy is ~70 kJ/mol, of which 50–60 kJ/mol stems from specific PI(3)P-interactions. By AT-MD, we could partition the binding mode into two types: (i) adhesion by electrostatic FYVE-PI(3)P interaction, and (ii) insertion of amphipathic loops. The AT simulations also demonstrated flexibility within the FYVE homodimer between the C-terminal heads and coiled-coil stem. This leads to a dynamic model whereby the 200 nm long coiled coil attached to the FYVE domain dimer can amplify local hinge-bending motions such that the Rab5-binding domain at the other end of the coiled coil can explore an area of 0.1 μm² in the search for a second endosome with which to interact.     

Thermal stability of single‐domain antibodies estimated by molecular dynamics simulations

Single‐domain antibodies (sdAbs) function like regular antibodies, however, consist of only one domain. Because of their low molecular weight, sdAbs have advantages with respect to production and delivery to their targets and for applications such as antibody drugs and biosensors. Thus, sdAbs with high thermal stability are required. In this work, we chose seven sdAbs, which have a wide range of melting temperature (T_m) values and known structures. We applied molecular dynamics (MD) simulations to estimate their relative stability and compared them with the experimental data. High‐temperature MD simulations at 400 K and 500 K were executed with simulations at 300 K as a control. The fraction of native atomic contacts, Q, measured for the 400 K simulations showed a fairly good correlation with the T_m values. Interestingly, when the residues were classified by their hydrophobicity and size, the Q values of hydrophilic residues exhibited an even better correlation, suggesting that stabilization is correlated with favorable interactions of hydrophilic residues. Measuring the Q value on a per‐residue level enabled us to identify residues that contribute significantly to the instability and thus demonstrating how our analysis can be used in a mutant case study.     

Structural, stability, dynamic and binding properties of the ALS-causing T46I mutant of the hVAPB MSP domain as revealed by NMR and MD simulations

T46I is the second mutation on the hVAPB MSP domain which was recently identified from non-Brazilian kindred to cause a familial amyotrophic lateral sclerosis (ALS). Here using CD, NMR and molecular dynamics (MD) simulations, we characterized the structure, stability, dynamics and binding capacity of the T46I-MSP domain. The results reveal: 1) unlike P56S which we previously showed to completely eliminate the native MSP structure, T46I leads to no significant disruption of the native secondary and tertiary structures, as evidenced from its far-UV CD spectrum, as well as Cα and Cβ NMR chemical shifts. 2) Nevertheless, T46I does result in a reduced thermodynamic stability and loss of the cooperative urea-unfolding transition. As such, the T46I-MSP domain is more prone to aggregation than WT at high protein concentrations and temperatures in vitro, which may become more severe in the crowded cellular environments. 3) T46I only causes a 3-fold affinity reduction to the Nir2 peptide, but a significant elimination of its binding to EphA4. 4) EphA4 and Nir2 peptide appear to have overlapped binding interfaces on the MSP domain, which strongly implies that two signaling networks may have a functional interplay in vivo. 5) As explored by both H/D exchange and MD simulations, the MSP domain is very dynamic, with most loop residues and many residues on secondary structures highly fluctuated or/and exposed to bulk solvent. Although T46I does not alter overall dynamics, it does trigger increased dynamics of several local regions of the MSP domain which are implicated in binding to EphA4 and Nir2 peptide. Our study provides the structural and dynamic understanding of the T46I-causing ALS; and strongly highlights the possibility that the interplay of two signaling networks mediated by the FFAT-containing proteins and Eph receptors may play a key role in ALS pathogenesis.     

Effects of PI(4,5)P2 concentration on the F-BAR domain membrane binding as revealed by coarse-grained simulations

Bin/Amphyphysin/Rvs (BAR) domain proteins form a key link between membrane remodeling and cytoskeleton dynamics. They are dimers that bind to membranes via electrostatic interactions with different preferences toward negatively charged lipids. In the present article, we examine the interactions of the F-BAR domain of nervous wreck (Nwk) with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂)-containing membranes using coarse-grained molecular dynamics. We demonstrated PI(4,5)P₂ concentration effects, identified the sequence of events that underlies the protein binding and identified amino acids involved in protein–lipid interactions. Our simulations point out the primary role of the basic stretch at the tips of the dimer, which anchors the protein to the membrane and initiates the binding process. When the PI(4,5)P₂ concentration is high, the protein stably associates with the membrane by its concave surface or by the opposite side. At low PI(4,5)P₂ concentration, the former orientation becomes more favorable; also a state with only one tip bound is observed, due to the weaker attachment and more pronounced association/dissociation events. Our results provide a theoretical model that describes the lipid-binding behavior of Nwk observed in vitro.     

Characteristic domain motion in the ribosome recycling factor revealed by 15N NMR relaxation experiments and molecular dynamics simulations

The backbone dynamics of ribosome recycling factor (RRF) from Escherichia coli in water were characterized by (15)N NMR relaxation analysis and molecular dynamics (MD) simulation. RRF is composed of two domains connected by a joint region that consists of two peptide chains, such that the overall structure seems to mimic that of tRNA. MD trajectories indicated that the relative orientation of domains varies on the nanosecond time scale. We analyzed the observed (15)N T(1), T(2), and NOE using an extended model-free spectral density function in which the domain motions with a nanosecond time scale were considered. At 30 degrees C, the order parameters of slow motion () were determined to be approximately 0.9 for domain I and 0.7 for domain II, respectively. These values indicate that domain I is nearly fixed on the molecular diffusion frame, and domain II is wobbling in a cone for which the semi-angle is about 30 degrees.     

Assembly of lipoprotein particles revealed by coarse-grained molecular dynamics simulations

High-density lipoproteins (HDL) function as cholesterol transporters, facilitating the removal of excess cholesterol from the body. Due to the heterogeneity of native HDL particles (both in size and shape), the details on how these protein-lipid particles form and the structure they assume in their lipid-associated states are not well characterized. We report here a study of the self-assembly of discoidal HDL particles using coarse-grained (CG) molecular dynamics. The microsecond simulations reveal the self-assembly of HDL particles from disordered protein-lipid complexes to form structures containing many of the features of the generally accepted double-belt model for discoidal HDL particles. HDL assembly is found to proceed in two broad steps, aggregation of proteins and lipids driven by the hydrophobic effect which occurs on a approximately 1 micros time scale, followed by the optimization of the protein structure driven by increasingly specific protein-protein interactions.     

Mutant bovine odorant-binding protein: Temperature affects the protein stability and dynamics as revealed by infrared spectroscopy and molecular dynamics simulations

Bovine odorant-binding protein (bOBP), a member of the lipocalin family, presents the so-called 3D "domain-swapped" protein structure. In fact, in solution, it appears as a dimer in which each monomer is composed by the classical lipocalin fold, with a central beta-barrel followed by a stretch of residues and the alpha-helix domain protruding out of the barrel and crossing the dimer interface. Recently, a deswapped mutant form of bOBP was obtained, in which a Gly residue was inserted after position 121 and the two residues in position 64 and 156 were replaced by Cys residues for restoring the disulfide bridge common to the lipocalin family. In this work, we used Fourier transform infrared spectroscopy and molecular dynamics simulations to investigate the effect of temperature on the structural stability and conformational dynamics of the mutant bOBP. The spectroscopic and molecular simulation data pointed out that the hydrophobic regions of the protein matrix appear to be an important factor for the protein stability and integrity. In addition, it was also found that the mutant bOBP is significantly stabilized by the binding of the ligand, which may have an impact on the biological function of bOBP. The obtained results will allow for a better use of this protein as probe for the design of advanced protein-based biosensors for the detection of compounds used in the fabrication of explosive powders.     

Experimental evaluation of single‐domain antibodies predicted by molecular dynamics simulations to have elevated thermal stability

Recently Bekker et al. [Bekker G‐J et al. Protein Sci. 2019;28:429–438.] described a computational strategy of applying molecular‐dynamics simulations to estimate the relative stabilities of single‐domain antibodies, and utilized their method to design changes with the aim of increasing the stability of a single‐domain antibody with a known crystal structure. The structure from which they generated potentially stabilizing mutations is an anti‐cholera toxin single domain antibody selected from a naïve library which has relatively low thermal stability, reflected by a melting point of 48°C. Their work was purely theoretical, so to examine their predictions, we prepared the parental and predicted stabilizing mutant single domain antibodies and examined their thermal stability, ability to refold and affinity. We found that the mutation that improved stability the most (~7°C) was one which changed an amino acid in CDR1 from an asparagine to an aspartic acid. This change unfortunately was also accompanied by a reduction in affinity. Thus, while their modeling did appear to successfully predict stabilizing mutations, introducing mutations in the binding regions is problematic. Of further interest, the mutations selected via their high temperature simulations, did improve refolding, suggesting that they were successful in stabilizing the structure at high temperatures and thereby decrease aggregation. Our result should permit them to reassess and refine their model and may one day lead to a usefulin silico approach to protein stabilization.     

Epigenetic gambling and epigenetic drift as an antagonistic pleiotropic mechanism of aging

Generations of biogerontologists have been puzzled by the marked intraspecific variations in lifespan of their experimental model organisms despite all efforts to control both genotype and environment. The most cogent example comes from life table studies of wild‐type Caenorhabditis elegans when grown in suspension cultures using axenic media. While nuclear and mitochondrial somatic mutations and ‘thermodynamic noise’ likely contribute to such lifespan variegations, I raise an additional hypothetical mechanism, one that may have evolved as a mechanism of phenotypic variation which could have preceded the evolution of meiotic recombination. I suggest that random changes in cellular gene expression (cellular epigenetic gambling or bet hedging) evolved as an adaptive mechanism to ensure survival of members of a group in the face of unpredictable environmental challenges. Once activated, it could lead to progressive epigenetic variegation (epigenetic drift) amongst all members of the group. Thus, while particular patterns of gene expression would be adaptive for a subset of reproductive individuals within a population early in life, once initiated, I predict that continued epigenetic drift will result in variable onsets and patterns of pathophysiology – perhaps yet another example of antagonistic pleiotropic gene action in the genesis of senescent phenotypes. The weakness of this hypothesis is that we do not currently have a plausible molecular mechanism for the putative genetic ‘randomizer’ of epigenetic expression, particularly one whose ‘setting’ may be responsive to the ecology in which a given species evolves. I offer experimental approaches, however, to search for the elusive epigenetic gambler(s).     

Autotriploid origin of Carassius auratus as revealed by chromosomal locus analysis

In the Dongting water system, the Carassius auratus (Crucian carp) complex is characterized by the coexistence of diploid forms (2n=100, 2nCC) and polyploid forms. Chromosomal and karyotypic analyses have suggested that the polyploid C. auratus has a triploid (3n=150, 3nCC) and a tetraploid origin (4n=200), respectively. However, there is a lack of direct genetic evidence to support this conclusion. In this paper, analysis of the 5S rDNA chromosomal locus revealed that the 3nCC is of triploid origin. Analysis of the species-specific chromosomal centromere locus revealed that 3nCC individuals possess three sets of C. auratus-derived chromosomes. Our results provide direct cytogenetic evidence suggesting that individuals with 150 chromosomes are of autotriploid origin within the C. auratus complex. It marks an important contribution to the study of polyploidization and the evolution of vertebrates.     

Structure of spheroidal HDL particles revealed by combined atomistic and coarse-grained simulations

Spheroidal high-density lipoprotein (HDL) particles circulating in the blood are formed through an enzymatic process activated by apoA-I, leading to the esterification of cholesterol, which creates a hydrophobic core of cholesteryl ester molecules in the middle of the discoidal phospholipid bilayer. In this study, we investigated the conformation of apoA-I in model spheroidal HDL (ms-HDL) particles using both atomistic and coarse-grained molecular dynamics simulations, which are found to provide consistent results for all HDL properties we studied. The observed small contribution of cholesteryl oleate molecules to the solvent-accessible surface area of the entire ms-HDL particle indicates that palmitoyloleoylphosphatidylcholines and apoA-I molecules cover the hydrophobic core comprised of cholesteryl esters particularly well. The ms-HDL particles are found to form a prolate ellipsoidal shape, with sizes consistent with experimental results. Large rigid domains and low mobility of the protein are seen in all the simulations. Additionally, the average number of contacts of cholesteryl ester molecules with apoA-I residues indicates that cholesteryl esters interact with protein residues mainly through their cholesterol moiety. We propose that the interaction of annular cholesteryl oleate molecules contributes to apoA-I rigidity stabilizing and regulating the structure and function of the ms-HDL particle.     

Structure and interactions of RecA: plasticity revealed by molecular dynamics simulations

Eleven independent simulations, each involving three consecutive molecules in the RecA filament, carried out on the protein from Mycobacterium tuberculosis, Mycobacterium smegmatis and Escherichia coli and their Adenosine triphosphate (ATP) complexes, provide valuable information which is complementary to that obtained from crystal structures, in addition to confirming the robust common structural framework within which RecA molecules from different eubacteria function. Functionally important loops, which are largely disordered in crystal structures, appear to adopt in each simulation subsets of conformations from larger ensembles. The simulations indicate the possibility of additional interactions involving the P-loop which remains largely invariant. The phosphate tail of the ATP is firmly anchored on the loop while the nucleoside moiety exhibits substantial structural variability. The most important consequence of ATP binding is the movement of the ‘switch’ residue. The relevant simulations indicate the feasibility of a second nucleotide binding site, but the pathway between adjacent molecules in the filament involving the two nucleotide binding sites appears to be possible only in the mycobacterial proteins.