Allosteric Interactions in Human Cytochrome P450 CYP3A4: The Role of Phenylalanine 213.

The role of Phe213 in the allosteric mechanism of human cytochrome P450 CYP3A4 was studied using a combination of progesterone (PGS) and carbamazepine (CBZ) as probe substrates. We expressed, purified, and incorporated into POPC Nanodiscs three mutants, F213A, F213S, and F213Y, and compared them with wild-type (WT) CYP3A4 by monitoring spectral titration, the rate of NADPH oxidation, and steady-state product turnover rates with pure substrates and substrate mixtures. All mutants demonstrated higher activity with CBZ, lower activity with PGS, and a reduced level of activation of CBZ epoxidation by PGS, which was most pronounced in the F213A mutant. Using all-atom molecular dynamics simulations, we compared the dynamics of WT CYP3A4 and the F213A mutant incorporated into the lipid bilayer and the effect of the presence of the PGS molecule at the allosteric peripheral site and evaluated the critical role of Phe213 in mediating the heterotropic allosteric interactions in CYP3A4.

Coarse-grained molecular simulations of allosteric cooperativity.

Interactions between a protein and a ligand are often accompanied by a redistribution of the population of thermally accessible conformations. This dynamic response of the protein's functional energy landscape enables a protein to modulate binding affinities and control binding sensitivity to ligand concentration. In this paper, we investigate the structural origins of binding affinity and allosteric cooperativity of binding two Ca(2+) ions to each domain of Calmodulin (CaM) through simulations of a simple coarse-grained model. In this model, the protein's conformational transitions between open and closed conformational ensembles are simulated explicitly and ligand binding and unbinding are treated implicitly within the grand canonical ensemble. Ligand binding is cooperative because the binding sites are coupled through a shift in the dominant conformational ensemble upon binding. The classic Monod-Wyman-Changeux model of allostery with appropriate binding free energies to the open and closed ensembles accurately describes the simulated binding thermodynamics. The simulations predict that the two domains of CaM have distinct binding affinity and cooperativity. In particular, the C-terminal domain binds Ca(2+) with higher affinity and greater cooperativity than the N-terminal domain. From a structural point of view, the affinity of an individual binding loop depends sensitively on the loop's structural compatibility with the ligand in the bound ensemble, as well as the conformational flexibility of the binding site in the unbound ensemble.

Comparing allosteric transitions in the domains of calmodulin through coarse-grained simulations.

Calmodulin (CaM) is a ubiquitous Ca(2+)-binding protein consisting of two structurally similar domains with distinct stabilities, binding affinities, and flexibilities. We present coarse grained simulations that suggest that the mechanism for the domain's allosteric transitions between the open and closed conformations depends on subtle differences in the folded state topology of the two domains. Throughout a wide temperature range, the simulated transition mechanism of the N-terminal domain (nCaM) follows a two-state transition mechanism while domain opening in the C-terminal domain (cCaM) involves unfolding and refolding of the tertiary structure. The appearance of the unfolded intermediate occurs at a higher temperature in nCaM than it does in cCaM consistent with nCaM's higher thermal stability. Under approximate physiological conditions, the simulated unfolded state population of cCaM accounts for 10% of the population with nearly all of the sampled transitions (approximately 95%) unfolding and refolding during the conformational change. Transient unfolding significantly slows the domain opening and closing rates of cCaM, which can potentially influence its Ca(2+)-binding mechanism.

Assessing the functional impact of protein binding site definition.

Many biomedical applications, such as classification of binding specificities or bioengineering, depend on the accurate definition of protein binding interfaces. Depending on the choice of method used, substantially different sets of residues can be classified as belonging to the interface of a protein. A typical approach used to verify these definitions is to mutate residues and measure the impact of these changes on binding. Besides the lack of exhaustive data, this approach also suffers from the fundamental problem that a mutation introduces an unknown amount of alteration into an interface, which potentially alters the binding characteristics of the interface. In this study we explore the impact of alternative binding site definitions on the ability of a protein to recognize its cognate ligand using a pharmacophore approach, which does not affect the interface. The study also shows that methods for protein binding interface predictions should perform above approximately F-score = 0.7 accuracy level to capture the biological function of a protein.

Glutamylation of Npm2 and Nap1 acidic disordered regions increases DNA mimicry and histone chaperone efficiency.

Histone chaperones-structurally diverse, non-catalytic proteins enriched with acidic intrinsically disordered regions (IDRs)-protect histones from spurious nucleic acid interactions and guide their deposition into and out of nucleosomes. Despite their conservation and ubiquity, the function of the chaperone acidic IDRs remains unclear. Here, we show that the Xenopus laevis Npm2 and Nap1 acidic IDRs are substrates for TTLL4 (Tubulin Tyrosine Ligase Like 4)-catalyzed post-translational glutamate-glutamylation. We demonstrate that to bind, stabilize, and deposit histones into nucleosomes, chaperone acidic IDRs function as DNA mimetics. Our biochemical, computational, and biophysical studies reveal that glutamylation of these chaperone polyelectrolyte acidic stretches functions to enhance DNA electrostatic mimicry, promoting the binding and stabilization of H2A/H2B heterodimers and facilitating nucleosome assembly. This discovery provides insights into both the previously unclear function of the acidic IDRs and the regulatory role of post-translational modifications in chromatin dynamics.

Assessing the functional impact of protein binding site definition.

Many biomedical applications, such as classification of binding specificities or bioengineering, depend on the accurate definition of protein binding interfaces. Depending on the choice of method used, substantially different sets of residues can be classified as belonging to the interface of a protein. A typical approach used to verify these definitions is to mutate residues and measure the impact of these changes on binding. Besides the lack of exhaustive data this approach generates, it also suffers from the fundamental problem that a mutation introduces an unknown amount of alteration into an interface, which potentially alters the binding characteristics of the interface. In this study we explore the impact of alternative binding site definitions on the ability of a protein to recognize its cognate ligand using a pharmacophore approach, which does not affect the interface. The study also provides guidance on the minimum expected accuracy of interface definition that is required to capture the biological function of a protein.

Glutamylation of Npm2 and Nap1 acidic disordered regions increases DNA charge mimicry to enhance chaperone efficiency.

Histone chaperones-structurally diverse, non-catalytic proteins enriched with acidic intrinsically disordered regions (IDRs)-protect histones from spurious nucleic acid interactions and guide their deposition into and out of nucleosomes. Despite their conservation and ubiquity, the function of the chaperone acidic IDRs remains unclear. Here, we show that the Xenopus laevis Npm2 and Nap1 acidic IDRs are substrates for TTLL4 (Tubulin Tyrosine Ligase Like 4)-catalyzed post-translational glutamate-glutamylation. We demonstrate that, to bind, stabilize, and deposit histones into nucleosomes, chaperone acidic IDRs function as DNA mimetics. Our biochemical, computational, and biophysical studies reveal that glutamylation of these chaperone polyelectrolyte acidic stretches functions to enhance DNA electrostatic mimicry, promoting the binding and stabilization of H2A/H2B heterodimers and facilitating nucleosome assembly. This discovery provides insights into both the previously unclear function of the acidic IDRs and the regulatory role of post-translational modifications in chromatin dynamics.

Computational Assessment of Protein-Protein Binding Specificity within a Family of Synaptic Surface Receptors.

Atomic-level information is essential to explain the formation of specific protein complexes in terms of structure and dynamics. The set of Dpr and DIP proteins, which play a key role in the neuromorphogenesis in the nervous system of Drosophila melanogaster, offer a rich paradigm to learn about protein-protein recognition. Many members of the DIP subfamily cross-react with several members of the Dpr family and vice versa. While there exists a total of 231 possible Dpr-DIP heterodimer complexes from the 21 Dpr and 11 DIP proteins, only 57 "cognate" pairs have been detected by surface plasmon resonance (SPR) experiments, suggesting that the remaining 174 pairs have low or unreliable binding affinity. Our goal is to assess the performance of computational approaches to characterize the global set of interactions between Dpr and DIP proteins and identify the specificity of binding between each DIP with their corresponding Dpr binding partners. In addition, we aim to characterize how mutations influence the specificity of the binding interaction. In this work, a wide range of knowledge-based and physics-based approaches are utilized, including mutual information, linear discriminant analysis, homology modeling, molecular dynamics simulations, Poisson-Boltzmann continuum electrostatics calculations, and alchemical free energy perturbation to decipher the origin of binding specificity of the Dpr-DIP complexes examined. Ultimately, the results show that those two broad strategies are complementary, with different strengths and limitations. Biological inter-relations are more clearly revealed through knowledge-based approaches combining evolutionary and structural features, the molecular determinants controlling binding specificity can be predicted accurately with physics-based approaches based on atomic models.

Cell fate decisions, transcription factors and signaling during early retinal development.

The development of the vertebrate eyes is a complex process starting from anterior-posterior and dorso-ventral patterning of the anterior neural tube, resulting in the formation of the eye field. Symmetrical separation of the eye field at the anterior neural plate is followed by two symmetrical evaginations to generate a pair of optic vesicles. Next, reciprocal invagination of the optic vesicles with surface ectoderm-derived lens placodes generates double-layered optic cups. The inner and outer layers of the optic cups develop into the neural retina and retinal pigment epithelium (RPE), respectively. In vitro produced retinal tissues, called retinal organoids, are formed from human pluripotent stem cells, mimicking major steps of retinal differentiation in vivo. This review article summarizes recent progress in our understanding of early eye development, focusing on the formation the eye field, optic vesicles, and early optic cups. Recent single-cell transcriptomic studies are integrated with classical in vivo genetic and functional studies to uncover a range of cellular mechanisms underlying early eye development. The functions of signal transduction pathways and lineage-specific DNA-binding transcription factors are dissected to explain cell-specific regulatory mechanisms underlying cell fate determination during early eye development. The functions of homeodomain (HD) transcription factors Otx2, Pax6, Lhx2, Six3 and Six6, which are required for early eye development, are discussed in detail. Comprehensive understanding of the mechanisms of early eye development provides insight into the molecular and cellular basis of developmental ocular anomalies, such as optic cup coloboma. Lastly, modeling human development and inherited retinal diseases using stem cell-derived retinal organoids generates opportunities to discover novel therapies for retinal diseases.

Gradient-driven diffusion and pattern formation in crowded mixtures.

Gradient-driven diffusion in crowded, multicomponent mixtures is a topic of high interest because of its role in biological processes such as transport in cell membranes. In partially phase-separated solutions, gradient-driven diffusion affects microstructure, which in turn affects diffusivity; a key question is how this complex coupling controls both transport and pattern formation. To examine these mechanisms, we study a two-dimensional multicomponent lattice gas model, where "tracer" molecules diffuse between a source and a sink separated by a solution of sticky "crowder" molecules that cluster to form dynamically evolving obstacles. In the high-temperature limit, crowders and tracers are miscible, and transport may be predicted analytically. At intermediate temperatures, crowders phase separate into clusters that drift toward the tracer sink. As a result, steady-state tracer diffusivity depends nonmonotonically on both temperature and crowder density, and we observe a variety of complex microstructures. In the low-temperature limit, crowders rapidly aggregate to form obstacles that are kinetically arrested; if crowder density is near the percolation threshold, resulting tracer diffusivity shows scaling behavior with the same scaling exponent as the random resistor network model. Though highly idealized, this simple model reveals fundamental mechanisms governing coupled gradient-driven diffusion, phase separation, and microstructural evolution in crowded mixtures.