Protonation-State Dependence of Hydration and Interactions in the Two Proton-Conducting Channels of Cytochrome c Oxidase.

Cytochrome c Oxidase (CcO), a membrane protein of the respiratory chain, pumps protons against an electrochemical gradient by using the energy of oxygen reduction to water. The ("chemical") protons required for this reaction and those pumped are taken up via two distinct channels, named D-channel and K-channel, in a step-wise and highly regulated fashion. In the reductive phase of the catalytic cycle, both channels transport protons so that the pumped proton passes the D-channel before the "chemical" proton has crossed the K-channel. By performing molecular dynamics simulations of CcO in the O→E redox state (after the arrival of the first reducing electron) with various combinations of protonation states of the D- and K-channels, we analysed the effect of protonation on the two channels. In agreement with previous work, the amount of water observed in the D-channel was significantly higher when the terminal residue E286 was not (yet) protonated than when the proton arrived at this end of the D-channel and E286 was neutral. Since a sufficient number of water molecules in the channel is necessary for proton transport, this can be understood as E286 facilitating its own protonation. K-channel hydration shows an even higher dependence on the location of the excess proton in the K-channel. Also in agreement with previous work, the K-channel exhibits a very low hydration level that likely hinders proton transfer when the excess proton is located in the lower part of the K-channel, that is, on the N-side of S365. Once the proton has passed S365 (towards the reaction site, the bi-nuclear centre (BNC)), the amount of water in the K-channel provides hydrogen-bond connectivity that renders proton transfer up to Y288 at the BNC feasible. No significant direct effect of the protonation state of one channel on the hydration level, hydrogen-bond connectivity, or interactions between protein residues in the other channel could be observed, rendering proton conductivity in the two channels independent of each other. Regulation of the order of proton uptake and proton passage in the two channels such that the "chemical" proton leaves its channel last must, therefore, be achieved by other means of communication, such as the location of the reducing electron.

Interaction of Thymine DNA Glycosylase with Oxidised 5-Methyl-cytosines in Their Amino- and Imino-Forms.

Thymine DNA Glycosylase (TDG) is an enzyme of the base excision repair mechanism and removes damaged or mispaired bases from DNA via hydrolysis of the glycosidic bond. Specificity is of high importance for such a glycosylase, so as to avoid the damage of intact DNA. Among the substrates reported for TDG are mispaired uracil and thymine but also formyl-cytosine and carboxyl-cytosine. Methyl-cytosine and hydroxylmethyl-cytosine are, in contrast, not processed by the TDG enzyme. We have in this work employed molecular dynamics simulations to explore the conformational dynamics of DNA carrying a formyl-cytosine or carboxyl-cytosine and compared those to DNA with the non-cognate bases methyl-cytosine and hydroxylmethyl-cytosine, as amino and imino tautomers. Whereas for the mispairs a wobble conformation is likely decisive for recognition, all amino tautomers of formyl-cytosine and carboxyl-cytosine exhibit the same Watson-Crick conformation, but all imino tautomers indeed form wobble pairs. The conformational dynamics of the amino tautomers in free DNA do not exhibit differences that could be exploited for recognition, and also complexation to the TDG enzyme does not induce any alteration that would indicate preferable binding to one or the other oxidised methyl-cytosine. The imino tautomers, in contrast, undergo a shift in the equilibrium between a closed and a more open, partially flipped state, towards the more open form upon complexation to the TDG enzyme. This stabilisation of the more open conformation is most pronounced for the non-cognate bases methyl-cytosine and hydroxyl-cytosine and is thus not a likely mode for recognition. Moreover, calculated binding affinities for the different forms indicate the imino forms to be less likely in the complexed DNA. These findings, together with the low probability of imino tautomers in free DNA and the indifference of the complexed amino tautomers, suggest that discrimination of the oxidised methyl-cytosines does not take place in the initial complex formation.

Hydrogen-Bonded Network and Water Dynamics in the D-channel of Cytochrome c Oxidase.

Proton transfer in cytochrome c oxidase (CcO) from the cellular inside to the binuclear redox centre as well as proton pumping through the membrane takes place through proton entrance via two distinct pathways, the D- and K-channel. Both channels show a dependence of their hydration level on the protonation states of their key residues, K362 for the K-channel, and E286 or D132 for the D-channel. In the oxidative half of CcO's catalytic cycle the D-channel is the proton-conducting path. For this channel, an interplay of protonation state of the D-channel residues with the water and hydrogen-bond dynamics has been observed in molecular dynamics simulations of the CcO protein, embedded in a lipid bi-layer, modelled in different protonation states. Protonation of residue E286 at the end of the D-channel results in a hydrogen-bonded network pointing from E286 to N139, that is against proton transport, and favouring N139 conformations which correspond to a closed asparagine gate (formed by residues N121 and N139). Consequently, the hydration level is lower than with unprotonated E286. In those models, the Asn gate is predominantly open, allowing water molecules to pass and thus increase the hydration level. The hydrogen-bonded network in these states exhibits longer life times of the Asn residues with water than other models and shows the D-channel to be traversable from the entrance, D132, to exit, E286. The D-channel can thus be regarded as auto-regulated with respect to proton transport, allowing proton passage only when required, that is the proton is located at the lower part of the D-channel (D132 to Asn gate) and not at the exit (E286).

Oxidation Enhances Binding of Extrahelical 5-Methyl-Cytosines by Thymine DNA Glycosylase.

The DNA repair protein thymine DNA glycosylase (TDG) removes mispaired or damaged bases, such as oxidized methyl-cytosine, from DNA by cleavage of the glycosidic bond between the sugar and the target base flipped into the enzyme's active site. The enzyme is active against formyl-cytosine and carboxyl-cytosine, whereas the lower oxidized hydroxymethyl-cytosine and methyl-cytosine itself are not processed by the enzyme. Molecular dynamics simulations with thermodynamic integration of TDG complexed to DNA carrying one of four different (oxidized) methyl-cytosine bases in extrahelcial conformation, methyl-cytosine (mC), hydroxymethyl-cytosine (hmC), formyl-cytosine (fC), or carboxyl-cytosine (caC), show a more favorable binding affinity of the higher oxidized forms, fC and caC, than the nonsubstrate bases hmC and mC. Despite rather comparable, reaction-competent conformations of the flipped bases in the active site of the enzyme, more and stronger interactions with active site residues account for the preferred binding of the higher oxidized bases. Binding of the negatively charged caC and the neutral fC are strengthened by interactions with positively charged His151. Our calculated proton affinities find this protonation state of His151 the preferred one in the presence of caC and conceivable in the presence of fC as well as increasing the binding affinity toward the two bases. Discrimination of the substrate bases is further achieved by the backbone of Tyr152 that forms a strong hydrogen bond to the carboxyl and formyl oxygen atoms of caC and fC, respectively, a contact that is completely lacking in mC and much weaker in hmC. Overall, our computational results indicate that the enzyme discriminates the different oxidation forms of methyl-cytosine already at the formation of the extrahelical complexes.

Molecular Dynamics Simulations of a Chimeric Androgen Receptor Protein (SPARKI) Confirm the Importance of the Dimerization Domain on DNA Binding Specificity.

The DNA binding domains of Androgen/Glucocorticoid receptors (AR/GR), members of class I steroid receptors, bind as a homo-dimer to a cis-regulatory element. These response elements are arranged as inverted repeat (IR) of hexamer "AGAACA", separated with a 3 base pairs spacer. DNA binding domains of the Androgen receptor, AR-DBDs, in addition, selectively recognize a direct-like repeat (DR) arrangement of this hexamer. A chimeric AR protein, termed SPARKI, in which the second zinc-binding motif of AR is swapped with that of GR, however, fails to recognize DR-like elements. By molecular dynamic simulations, we identify how the DNA binding domains of the wild type AR/GR, and also the chimeric SPARKI model, distinctly interact with both IR and DR response elements. AR binds more strongly to DR than GR binds to IR elements. A SPARKI model built from the structure of the AR (SPARKI-AR) shows significantly fewer hydrogen bond interactions in complex with a DR sequence than with an IR sequence. Moreover, a SPARKI model based on the structure of the GR (SPARKI-GR) shows a considerable distortion in its dimerization domain when complexed to a DR-DNA whereas it remains in a stable conformation in a complex with an IR-DNA. The diminished interaction of SPARKI-AR with and the instability of SPARKI-GR on DR response elements agree with SPARKI's lack of affinity for these sequences. The more GR-like binding specificity of the chimeric SPARKI protein is further emphasized by both SPARKI models binding even more strongly to IR elements than observed for the DNA binding domain of the GR.