Towards computational specificity screening of DNA-binding proteins.

DNA-binding proteins are key players in the regulation of gene expression and, hence, are essential for cell function. Chimeric proteins composed of DNA-binding domains and DNA modifying domains allow for precise genome manipulation. A key prerequisite is the specific recognition of a particular nucleotide sequence. Here, we quantitatively assess the binding affinity of DNA-binding proteins by molecular dynamics-based alchemical free energy simulations. A computational framework was developed to automatically set up in silico screening assays and estimate free energy differences using two independent procedures, based on equilibrium and non-equlibrium transformation pathways. The influence of simulation times on the accuracy of both procedures is presented. The binding specificity of a zinc-finger transcription factor to several sequences is calculated, and agreement with experimental data is shown. Finally we propose an in silico screening strategy aiming at the derivation of full specificity profiles for DNA-binding proteins.

Kinase-activity-independent functions of atypical protein kinase C in Drosophila.

Polarity of many cell types is controlled by a protein complex consisting of Bazooka/PAR-3 (Baz), PAR-6 and atypical protein kinase C (aPKC). In Drosophila, the Baz-PAR-6-aPKC complex is required for the control of cell polarity in the follicular epithelium, in ectodermal epithelia and neuroblasts. aPKC is the main signaling component of this complex that functions by phosphorylating downstream targets, while the PDZ domain proteins Baz and PAR-6 control the subcellular localization and kinase activity of aPKC. We compared the mutant phenotypes of an aPKC null allele with those of four novel aPKC alleles harboring point mutations that abolish the kinase activity or the binding of aPKC to PAR-6. We show that these point alleles retain full functionality in the control of follicle cell polarity, but produce strong loss-of-function phenotypes in embryonic epithelia and neuroblasts. Our data, combined with molecular dynamics simulations, show that the kinase activity of aPKC and its ability to bind PAR-6 are only required for a subset of its functions during development, revealing tissue-specific differences in the way that aPKC controls cell polarity.

Molecular determinants of snurportin 1 ligand affinity and structural response upon binding.

The transport of large biomolecules such as proteins and RNA across nuclear pore complexes is a field of strong interest and research. Although the basic mechanisms are fairly well understood, the details of the underlying intermolecular interaction within these transport complexes are still unclear. The recognition dynamics and energetics of cargo binding to the transport receptor are not yet resolved. Here, the binding of dimethylated RNA-caps to snurportin 1 is studied by molecular-dynamics simulations. The simulations reveal a strong structural response of the protein upon RNA-cap release. In particular, major rearrangements occur in regions already intrinsically flexible in the holo structure. Additionally, the difference in free energy of binding to snurportin 1 between the two methylation states of the RNA-cap, responsible for the directionality of the transport is quantified. In particular, desolvation of the ligand is revealed as the key-step in binding to snurportin 1. These findings suggest that the binding of m(3)G-capped RNA is mainly driven by the enhanced water entropy gain of the solvation shell.

Accuracy and convergence of free energy differences calculated from nonequilibrium switching processes.

The molecular-dynamics-based calculation of accurate free energy differences for biomolecular systems is a challenging task. Accordingly, convergence and accuracy of established equilibrium methods has been subject of many studies, often focusing at small test systems. In contrast, the potential of more recently proposed nonequilibrium methods, derived from the Jarzynski and Crooks equalities, has not yet fully been explored. Here, we compare the performance of these methods by calculating free energy differences for test systems at different levels of complexity and varying extent of the involved perturbations. We consider the interconversion of ethane into methanol, the switching of a tryptophane-sidechain in a tripeptide, and the binding of two different ligands to the globular protein snurportin 1. On the basis of our results, we suggest and assess a new nonequilibrium free energy method, Crooks Gaussian Intersection (CGI), which combines the advantages of existing methods. CGI is highly parallelizable and, for the test systems considered here, is shown to outperform the other studied equilibrium and nonequilibrium methods.

Ultrafast deactivation of an excited cytosine-guanine base pair in DNA.

Multiconfigurational ab initio calculations and QM/MM molecular dynamics simulations of a photoexcited cytosine-guanine base pair in both gas phase and embedded in the DNA provide detailed structural and dynamical insights into the ultrafast radiationless deactivation mechanism. Photon absorption promotes transfer of a proton from the guanine to the cytosine. This proton transfer is followed by an efficient radiationless decay of the excited state via an extended conical intersection seam. The optimization of the conical intersection revealed that it has an unusual topology, in that there is only one degeneracy-lifting coordinate. This is the central mechanistic feature for the decay both in vacuo and in the DNA. Radiationless decay occurs along an extended hyperline nearly parallel to the proton-transfer coordinate, indicating the proton transfer itself is not directly responsible for the deactivation. The seam is displaced from the minimum energy proton-transfer path along a skeletal deformation of the bases. Decay can thus occur anywhere along the single proton-transfer coordinate, accounting for the remarkably short excited-state lifetime of the Watson-Crick base pair. In vacuo, decay occurs after a complete proton transfer, whereas in DNA, decay can also occur much earlier. The origin of this effect lies in the temporal electrostatic stabilization of dipole in the charge-transfer state in DNA.

Rearrangements in a hydrophobic core region mediate cAMP action in the regulatory subunit of PKA.

cAMP-dependent protein kinase (PKA) forms an inactive heterotetramer of two regulatory (R; with two cAMP-binding domains A and B each) and two catalytic (C) subunits. Upon the binding of four cAMP molecules to the R dimer, the monomeric C subunits dissociate. Based on sequence analysis of cyclic nucleotide-binding domains in prokaryotes and eukaryotes and on crystal structures of cAMP-bound R subunit and cyclic nucleotide-free Epac (exchange protein directly activated by cAMP), four amino acids were identified (Leu203, Tyr229, Arg239 and Arg241) and probed for cAMP binding to the R subunits and for R/C interaction. Arg239 and Arg241 (mutated to Ala and Glu) displayed no differences in the parameters investigated. In contrast, Leu203 (mutated to Ala and Trp) and Tyr229 (mutated to Ala and Thr) exhibited up to 30-fold reduced binding affinity for the C subunit and up to 120-fold reduced binding affinity for cAMP. Tyr229Asp showed the most severe effects, with 350-fold reduced affinity for cAMP and no detectable binding to the C subunit. Based on these results and structural data in the cAMP-binding domain, a switch mechanism via a hydrophobic core region is postulated that is comparable to an activation model proposed for Epac.