Inferring chromosome radial organization from Hi-C data.

BACKGROUND: The nonrandom radial organization of eukaryotic chromosome territories (CTs) inside the nucleus plays an important role in nuclear functional compartmentalization. Increasingly, chromosome conformation capture (Hi-C) based approaches are being used to characterize the genome structure of many cell types and conditions. Computational methods to extract 3D arrangements of CTs from this type of pairwise contact data will thus increase our ability to analyze CT organization in a wider variety of biological situations. RESULTS: A number of full-scale polymer models have successfully reconstructed the 3D structure of chromosome territories from Hi-C. To supplement such methods, we explore alternative, direct, and less computationally intensive approaches to capture radial CT organization from Hi-C data. We show that we can infer relative chromosome ordering using PCA on a thresholded inter-chromosomal contact matrix. We simulate an ensemble of possible CT arrangements using a force-directed network layout algorithm and propose an approach to integrate additional chromosome properties into our predictions. Our CT radial organization predictions have a high correlation with microscopy imaging data for various cell nucleus geometries (lymphoblastoid, skin fibroblast, and breast epithelial cells), and we can capture previously documented changes in senescent and progeria cells. CONCLUSIONS: Our analysis approaches provide rapid and modular approaches to screen for alterations in CT organization across widely available Hi-C data. We demonstrate which stages of the approach can extract meaningful information, and also describe limitations of pairwise contacts alone to predict absolute 3D positions.

Regulatory Mechanics of Constitutive Androstane Receptors: Basal and Ligand-Directed Actions.

Constitutive androstane receptor (CAR) is a nuclear hormone receptor that primarily functions in sensing and metabolizing xenobiotics. The basal activity of this receptor is relatively high, and CAR is deemed active in the absence of ligand. The (over)activation can promote drug toxicity and tumor growth. Thus, therapeutic treatments seek inverse agonists to inhibit or modulate CAR activities. To advance our understanding of the regulatory mechanisms of CAR, we used computational and experimental approaches to elucidate three aspects of CAR activation and inactivation: (1) ligand-dependent actions, (2) ligand-orthologue specificity, and (3) constitutive activity. For ligand-dependent actions, we examined the ligand-bound simulations and identified two sets of ligand-induced contacts promoting CAR activation via coactivator binding (H11-H12 contact) or inactivation via corepressor binding (H4-H11 contact). For orthologue specificity, we addressed a puzzling fact that murine CAR (mCAR) and human CAR (hCAR) respond differently to the same ligand (CITCO), despite their high sequence homology. We found that the helix H7 of hCAR is responsible for a stronger binding of the ligand CITCO compared to mCAR, hence a stronger CITCO-induced activation. For basal activity, we reported computer-generated unliganded CAR structures and critical mutagenesis (mCAR's V209A and N333D) results of a cell-based transcription assay. Our results reveal that the basal conformation of CAR shares prominent features with the agonist-bound form, and helix HX has an important contribution to the constitutive activity. These findings altogether can be useful for the understanding of constitutively active receptors and the design of drug molecules targeting them.

Brownian dynamics simulation of peptides with the University of Houston Brownian Dynamics (UHBD) program.

This chapter provides the background theory and a practical protocol for performing Brownian dynamics simulation of peptides. Brownian dynamics simulation represents a complementary approach to Monte Carlo and molecular dynamics methods. Unlike Monte Carlo methods, it could provide dynamical information in a timescale longer than the momentum relaxation time. On the other hand, it is faster than molecular dynamics by approximating the solvent by a continuum and by operating in the over-damped limit. This chapter introduces the use of the University of Houston Brownian Dynamics (UHBD) program [1, 2] to perform Brownian dynamics simulation on peptides.

Solvent-induced α- to 3(10)-helix transition of an amphiphilic peptide.

The amphiphilic peptide of the triacylglycerol lipase derived from Pseudomonas aeruginosa plays a critical role in guarding the gate for ligand access. Conformations of this peptide at several water-oil interfaces and in protein environments were compared using atomistic simulations with explicit solvents. In oil-containing solvents, this peptide is able to retain a folded structure. Interestingly, when the peptide is immersed in a low-polarity solvent environment, it exhibits a "coalesced" helix structure, which has both α- and 3(10)-helix components. The observation that the 3(10)-helical conformation is populated in a highly nonpolar environment is consistent with a previous report on polymethylalanine. Frequent interconversions of the secondary structure (between α-helix and 3(10)-helix) of the peptide are also observed. We further studied how this solvent-induced structural transition may be connected to the trigger mechanism of lipase gating and how the lipase senses the hydrophobic-hydrophilic interface.

High-pressure effect on the dynamics of solvated peptides.

The dynamics of peptides has a direct connection to how quickly proteins can alter their conformations. The speed of exploring the free energy landscape depend on many factors, including the physical parameters of the environment, such as pressure and temperature. We performed a series of molecular dynamics simulations to investigate the pressure-temperature effects on peptide dynamics, especially on the torsional angle and peptide-water hydrogen bonding (H-bonding) dynamics. Here, we show that the dynamics of the omega angle and the H-bonding dynamics between water and the peptide are affected by pressure. At high temperature (500 K), both the dynamics of the torsional angle ω and H-bonding slow down significantly with increasing pressure, interestingly, at approximately the same rate. However, at a lower temperature of 300 K, the observed trend on H-bonding dynamics as a function of pressure reverses, i.e., higher pressure speeds up H-bonding dynamics.

Subtype-specific conservation of isoleucine 309 in the envelope V3 domain is linked to immune evasion in subtype C HIV-1 infection.

The V3 region of the HIV-1 envelope (Env) glycoprotein gp120 is a key functional domain yet it exhibits distinct mutational patterns across subtypes. Here an invariant residue (Ile 309) was replaced with Leu in 7 subtype C patient-derived Envs from recent infection and 4 related neutralizing antibody escape variants that emerged later. For these 11 Envs, I309L did not alter replication in primary CD4 T cells; however, replication in monocyte-derived macrophages was enhanced. Infection of cell lines with low CD4 or CCR5 revealed that I309L enhanced utilization of CD4 but did not affect the ability to use CCR5. This CD4-enhanced phenotype tracked with sensitivity to sCD4, indicating increased exposure of the CD4 binding site. The results suggest that Ile 309 preserves a V3-mediated masking function that occludes the CD4 binding site. The findings point to an immune evasion strategy in subtype C Env to protect this vulnerable immune target.

Conformational switching upon phosphorylation: a predictive framework based on energy landscape principles.

We investigate how post-translational phosphorylation modifies the global conformation of a protein by changing its free energy landscape using two test proteins, cystatin and NtrC. We first examine the changes in a free energy landscape caused by phosphorylation using a model containing information about both structural forms. For cystatin the free energy cost is fairly large indicating a low probability of sampling the phosphorylated conformation in a perfectly funneled landscape. The predicted barrier for NtrC conformational transition is several times larger than the barrier for cystatin, indicating that the switch protein NtrC most probably follows a partial unfolding mechanism to move from one basin to the other. Principal component analysis and linear response theory show how the naturally occurring conformational changes in unmodified proteins are captured and stabilized by the change of interaction potential. We also develop a partially guided structure prediction Hamiltonian which is capable of predicting the global structure of a phosphorylated protein using only knowledge of the structure of the unphosphorylated protein or vice versa. This algorithm makes use of a generic transferable long-range residue contact potential along with details of structure short range in sequence. By comparing the results obtained with this guided transferable potential to those from the native-only, perfectly funneled Hamiltonians, we show that the transferable Hamiltonian correctly captures the nature of the global conformational changes induced by phosphorylation and can sample substantially correct structures for the modified protein with high probability.

Insight into the role of hydration on protein dynamics.

The potential energy surface of a protein is rough. This intrinsic energetic roughness affects diffusion, and hence the kinetics. The dynamics of a system undergoing Brownian motion on this surface in an implicit continuum solvent simulation can be tuned via the frictional drag or collision frequency to be comparable to that of experiments or explicit solvent simulations. We show that the kinetic rate constant for a local rotational isomerization in stochastic simulations with continuum solvent and a collision frequency of 2 ps(-1) is about 10(4) times faster than that in explicit water and experiments. A further increase in the collision frequency to 60 ps(-1) slows down the dynamics, but does not fully compensate for the lack of explicit water. We also show that the addition of explicit water does not only slow down the dynamics by increasing the frictional drag, but also increases the local energetic roughness of the energy landscape by as much as 1.0 kcal/mol.

Elasticity of peptide omega bonds.

We calculated the changes of the free energy profile of the peptidyl-prolyl torsional angle of the dipeptide valine-proline under pulling forces by simulations. Using a dynamic model built on the equilibrium properties of this system and previously studied dynamic properties of cis-trans isomerization of other dipeptides, we calculated the dynamic viscoelasticity of this degree of freedom. The results show significant differences between how thermal and mechanical forces alter the equilibrium and the dynamics of the isomerization transition. The former does not change the barrier heights but changes the prefactor of the kinetics owing to temperature effects, while the latter changes minima and thus the population. The force that is required to "excite" this degree of freedom is small. Compared to other systems, we found that this degree of freedom becomes already quite rigid at several hertz, which is a much lower value due to the high barrier of the cis-trans isomerization. We also found that the tensile elastic modulus of densely packed omega bonds is at the order of GPa, which is comparable to that of polymer materials. These results give mechanical properties of polyproline elasticity of a local nature and provide guidance for future experimental designs.

Brownian dynamics simulation of helix-capping motifs.

Helix-capping motifs are believed to play an important role in stabilizing alpha-helices and defining helix start and stop signals. We performed microsecond scale Brownian dynamics simulations to study ten XAAD sequences, with X = (A,E,I,L,N,Q,S,T,V,Y), to examine their propensity to form helix capping motifs and correlate these results with those obtained from analyzing a structural database of proteins. For the widely studied capping box motif S**D, where the asterisk can be any amino acid residue, the simulations suggested that one of the two hydrogen bonds proposed earlier as a stabilizing factor might not be as important. On the other hand, side-chain interactions between the capping residue and the third residue downstream on the polypeptide chain might also play a role in stabilizing this motif. These results are consistent with explicit-solvent molecular dynamics simulations of two capping box motifs found in the proteins BPTI and alpha-dendrotoxin. Principal component analysis of the SAAD trajectory showed that the first three principal components, after those corresponding to translational-rotational motion were removed, accounted for more than half of the conformational fluctuations. The first component separated the conformational space into two parts with the all-helical conformation and the capping box motif lying largely in one part. The second component, on the other hand, could be used to describe conformational transitions between the all-helical form and the capping box motif.