Location and functional significance of retinol-binding sites on the serine/threonine kinase, c-Raf.

Redox activations of serine/threonine kinases represent alternate pathways in which vitamin A plays a crucial co-factor role. Vitamin A binds the zinc finger domain of c-Raf with nanomolar affinity. The retinoid-binding site has been mapped within this structure by scanning mutagenesis. The deduced contact sites were found anchored on Phe-8, counting from the 1st conserved histidine of the zinc finger. These sites agreed with contact amino acids identified by computational docking. The boundaries of a related binding pocket were identified by mutagenesis and partially confirmed by docking trials in the protein kinase C-alpha C1A zinc finger. They comprised Phe-7, Phe-8, and Trp-22. This trio was absent from the alphaC1B domain, explaining why the latter did not bind retinol. Reconfiguring at a minimum the two corresponding amino acids of alphaC1B, Thr-7 and Tyr-22, to conform to alphaC1A converted this domain to a binder. Deletion of the predicted retinoid-binding site in the full-length molecule created a mutant c-Raf that was deficient in retinol-dependent redox activation but fully responsive to epidermal growth factor. Our findings indicate that ligation of retinol to a specific site embedded in the regulatory domain is an important feature of c-Raf regulation in the redox pathway.

Shorter side chains optimize helix-helix packing.

A systematic study of helix-helix packing in a comprehensive database of protein structures revealed that the side chains inside helix-helix interfaces on average are shorter than those in the noninterface parts of the helices. The study follows our earlier study of this effect in transmembrane helices. The results obtained on the entire database of protein structures are consistent with those obtained on the transmembrane helices. The difference in the length of interface and noninterface side chains is small but statistically significant. It indicates that helices, if viewed along their main axis, statistically are not circular, but have a flattened interface. This effect brings the helices closer to each other and creates a tighter structural packing. The results provide an interesting insight into the aspects of protein structure and folding.

The role of geometric complementarity in secondary structure packing: a systematic docking study.

A strong similarity between the major aspects of protein folding and protein recognition is one of the emerging fundamental principles in protein science. A crucial importance of steric complementarity in protein recognition is a well-established fact. The goal of this study was to assess the importance of the steric complementarity in protein folding, namely, in the packing of the secondary structure elements. Although the tight packing of protein structures, in general, is a well-known fact, a systematic study of the role of geometric complementarity in the packing of secondary structure elements has been lacking. To assess the role of the steric complementarity, we used a docking procedure to recreate the crystallographically determined packing of secondary structure elements in known protein structures by using the geometric match only. The docking results revealed a significant percentage of correctly predicted packing configurations. Different types of pairs of secondary structure elements showed different degrees of steric complementarity (from high to low: beta-beta, loop-loop, alpha-alpha, and alpha-beta). Interestingly, the relative contribution of the steric match in different types of pairs was correlated with the number of such pairs in known protein structures. This effect may indicate an evolutionary pressure to select tightly packed elements of secondary structure to maximize the packing of the entire structure. The overall conclusion is that the steric match plays an essential role in the packing of secondary structure elements. The results are important for better understanding of principles of protein structure and may facilitate development of better methods for protein structure prediction.

DNA binding is required for the apoptogenic action of apoptosis inducing factor.

The execution of apoptosis or programmed cell death comprises both caspase-dependent and caspase-independent processes. Apoptosis inducing factor (AIF) was identified as a major player in caspase-independent cell death. It induces chromatin condensation and initial DNA cleavage via an unknown molecular mechanism. Here we report the crystal structure of human AIF at 1.8 A resolution. The structure reveals the presence of a strong positive electrostatic potential at the AIF surface, although the calculated isoelectric point for the entire protein is neutral. We show that recombinant AIF interacts with DNA in a sequence-independent manner. In addition, in cells treated with an apoptotic stimulus, endogenous AIF becomes co-localized with DNA at an early stage of nuclear morphological changes. Structure-based mutagenesis shows that DNA-binding defective mutants of AIF fail to induce cell death while retaining nuclear translocation. The potential DNA-binding site identified from mutagenesis also coincides with computational docking of a DNA duplex. These observations suggest that AIF-induced nuclear apoptosis requires a direct interaction with DNA.

Theoretical Study of the Diels-Alder Reaction of Selenoaldehydes and Selenoketones.

The Diels-Alder reaction of a series of substituted selenoaldehydes (Se=CHR; R = H, F, Me, OMe, CH(2)F, CF(3), and CN) or selenoketones (Se=CR(2); R = Me and CN) with 1,3-butadiene or 2-methoxy-1,3-butadiene to yield 3,6-dihydro-2H-selenopyrans were examined using B3LYP with a modified 6-31G basis set. This method is compared with results from a number of standard ab initio procedures and compares well with post-HF results. The Diels-Alder reaction of the selenocarbonyl compounds proceeds through a concerted, though asynchronous, transition state. Strong electron-withdrawing groups alter the mechanism; a charge-transfer complex is first formed, followed by a concerted TS before reaching the heterocyclic product. The transition state geometry, regiochemistry, and dependence of the activation barrier on substituents can be understood in terms of FMO theory.

Ab Initio Study of the Thio-Ene Reaction. 1. The Enophile Substituent Effect.

The ene reaction between propene and various enophiles (ethene, methanal, methanethial, ethanethial, and cyanomethanethial) were examined at the MP4/6-31G//MP2/6-31G level. The transition structures are all cyclic and concerted. The activation barriers for the thiocarbonyls are significantly lower (ranging from 16.0 to 25.0 kcal mol(-1)) than for ethene (36.3 kcal mol(-1)) or methanal 31.1 kcal mol(-1)). This trend, along with the trend in activation energies among the substituted thials, is completely consistent with FMO arguments. Comparison with experiment, particularly the thiol vs sulfide production, is also in complete agreement.