Previous studies have shown that the unusually long S5-P linker lining human ether a-go-go related gene's (hERG's) outer vestibule is critical for its channel function: point mutations at high-impact positions here can interfere with the inactivation process and, in many cases, also reduce the pore's K+ selectivity. Because no data are available on the equivalent region in the available K channel crystal structures to allow for homology modeling, we used alternative approaches to model its three-dimensional structure. The first part of this article describes mutant cycle analysis used to identify residues on hERG's outer vestibule that interact with specific residues on the interaction surface of BeKm-1, a peptide toxin with known NMR structure and a high binding affinity to hERG. The second part describes molecular modeling of hERG's pore domain. The transmembrane region was modeled after the crystal structure of KvAP pore domain. The S5-P linker was docked to the transmembrane region based on data from previous NMR and mutagenesis experiments, as well as a set of modeling criteria. The models were further restrained by contact points between hERG's outer vestibule and the bound BeKm-1 toxin molecule deduced from the mutant cycle analysis. Based on these analyses, we propose a working model for the open conformation of the outer vestibule of the hERG channel, in which the S5-P linkers interact with the pore loops to influence ion flux through the pore.

Studies on voltage-gated K channels such as Shaker have shown that positive charges in the voltage-sensor (S4) can form salt bridges with negative charges in the surrounding transmembrane segments in a state-dependent manner, and different charge pairings can stabilize the channels in closed or open states. The goal of this study is to identify such charge interactions in the hERG channel. This knowledge can provide constraints on the spatial relationship among transmembrane segments in the channel's voltage-sensing domain, which are necessary for modeling its structure. We first study the effects of reversing S4's positive charges on channel activation. Reversing positive charges at the outer (K525D) and inner (K538D) ends of S4 markedly accelerates hERG activation, whereas reversing the 4 positive charges in between either has no effect or slows activation. We then use the 'mutant cycle analysis' to test whether D456 (outer end of S2) and D411 (inner end of S1) can pair with K525 and K538, respectively. Other positive charges predicted to be able, or unable, to interact with D456 or D411 are also included in the analysis. The results are consistent with predictions based on the distribution of these charged residues, and confirm that there is functional coupling between D456 and K525 and between D411 and K538.

Conformational preferences of modified nucleoside, N(4)-acetylcytidine, ac(4)C have been investigated using quantum chemical semi-empirical RM1 method. Automated geometry optimization using PM3 method along with ab initio methods HF SCF (6-31G**), and density functional theory (DFT; B3LYP/6-31G**) have also been made to compare the salient features. The most stable conformation of N(4)-acetyl group of ac(4)C prefers "proximal" orientation. This conformation is stabilized by intramolecular hydrogen bonding between O(7)···HC(5), O(2)···HC2', and O4'···HC(6). The "proximal" conformation of N(4)-acetyl group has also been observed in another conformational study of anticodon loop of E. coli elongator tRNA(Met). The solvent accessible surface area (SASA) calculations revealed the role of ac(4)C in anticodon loop. The explicit molecular dynamics simulation study also shows the "proximal" orientation of N(4)-acetyl group. The predicted "proximal" conformation would allow ac(4)C to interact with third base of codon AUG/AUA whereas the 'distal' orientation of N(4)-acetyl cytidine side-chain prevents such interactions. Single point energy calculation studies of various models of anticodon-codon bases revealed that the models ac(4)C(34)(Proximal):G3, and ac(4)C(34)(Proximal):A3 are energetically more stable as compared to models ac(4)C(34)(Distal):G3, and ac(4)C(34)(Distal):A3, respectively. MEPs calculations showed the unique potential tunnels between the hydrogen bond donor-acceptor atoms of ac(4)C(34)(Proximal):G3/A3 base pairs suggesting role of ac(4)C in recognition of third letter of codons AUG/AUA. The "distal" conformation of ac(4)C might prevent misreading of AUA codon. Hence, this study could be useful to understand the role of ac(4)C in the tertiary structure folding of tRNA as well as in the proper recognition of codons during protein biosynthesis process.

Conformational preferences of the base substituent in hypermodified nucleotide queuosine 5'-monophosphate 'pQ' and its protonated form 'pQH+' have been studied using quantum chemical Perturbative Configuration Interaction with Localized Orbitals PCILO method. The salient points have also been examined using molecular mechanics force field MMFF, parameterized modified neglect of differential overlap PM3 and Hartree Fock-Density Functional Theory HF DFT (pBP/DN*) approaches. Aqueous solvation of pQ and pQH+ has also been studied using molecular dynamics simulations. Consistent with the observed crystal structure, in isolated protonated form pQH+, the quaternary amine HN(13)(+)H, of the sidechain having 7-aminomethyl linkage, hydrogen bonds with the carbonyl oxygen O(10) of the base. However, N(13)H-O(10) hydrogen bonding is not preferred for unprotonated pQ, whether isolated or hydrated. Interaction between the 5'-phosphate and the 7-aminomethyl group is more likely for isolated pQ. The cyclopentenediol hydroxyl group O4"H may hydrogen bond with the O(10) in isolated pQ as well as in pQH+. The O4"H may hydrogen bond with the 5'-phosphate as well. The presence of -CH2-NH- and O"H groups in pQ and pQH+ allows interesting possibilities for intranucleotide hydrogen bonds and interactions across the anticodon loop. Simultaneous hydrogen bonds O2P-HN(13)+H-O(10) are indicated for hydrated pQH+. Unlike weak involvement of O4"H, these interactions also persist in hydrated pQH+ and may much reduce backbone flexibility. Resulting sub-optimal Q:C base pairing leads to unbiased reading of U or C as the third codon letter. Cyclopentenediol hydroxyl groups may interact with other biomolecules, allowing specific recognition. Prospective pQ(34) and pQ(34)H+ sites for codon-anticodon base pairing remain unhindered, but non canonical Q:G base pairing (amber-suppression) is ruled out.

Conformational preferences of the modified nucleosides N(2)-methylguanosine (m(2)G) and N(2), N(2)-dimethylguanosine (m(2)(2)G) have been studied theoretically by using quantum chemical perturbative configuration interaction with localized orbitals (PCILO) method. Automated complete geometry optimization using semiempirical quantum chemical RM1, along with ab initio molecular orbital Hartree-Fock (HF-SCF), and density functional theory (DFT) calculations has also been made to compare the salient features. Single-point energy calculation studies have been made on various models of m(2)G26:C/A/U44 and m(2)(2)G26:C/A/U44. The glycosyl torsion angle prefers "syn" (χ = 286°) conformation for m(2)G and m(2)(2)G molecules. These conformations are stabilized by N(3)-HC2' and N(3)-HC3' by replacing weak interaction between O5'-HC(8). The N(2)-methyl substituent of (m(2)G26) prefers "proximal" or s-trans conformation. It may also prefer "distal" or s-cis conformation that allows base pairing with A/U44 instead of C at the hinge region. Thus, N(2)-methyl group of m(2)G may have energetically two stable s-trans m(2)G:C/A/U or s-cis m(2)G:A/U rotamers. This could be because of free rotations around C-N bond. Similarly, N(2), N(2)-dimethyl substituent of (m(2)(2)G) prefers "distal" conformation that may allow base pairing with A/U instead of C at 44th position. Such orientations of m(2)G and m(2)(2)G could play an important role in base-stacking interactions at the hinge region of tRNA during protein biosynthesis process.

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Conformational preferences of hypermodified nucleoside, 4-amino-2-(N(6)-lysino)-1-(beta-D-ribofuranosyl) pyrimidinium (Lysidine or 2-lysyl cytidine), usually designated as k(2)C, have been investigated theoretically by the quantum chemical perturbative configuration interaction with localized orbitals (PCILO) method. The zwitterionic, non-zwitterionic, neutral, and tautomeric forms have been studied. Automated geometry optimization using molecular mechanics force field (MMFF), semi-empirical quantum chemical PM3, and ab initio molecular orbital Hartree-Fock SCF quantum mechanical calculations have also been made to compare the salient features. The predicted most stable conformations of zwitterionic, non-zwitterionic, neutral, and tautomeric form are such that in each of these molecules the orientation of lysidine moiety (R) is trans to the N(1) of cytidine. The preferred base orientation is anti (chi = 3 degrees ) and the lysine substituent folds back toward the ribose ring. This results in hydrogen bonding between the carboxyl oxygen O(12a) of lysine moiety and the 2'-hydroxyl group of ribose sugar. In all these four forms of lysidine O(12a)...H-C(9) and O(12b)...H-N(11) interactions provide stability to respective stable conformers. Watson-Crick base pairing of lysidine with A is feasible only with the tautomeric form of usual anti oriented lysidine. This can help in recognition of AUA codon besides in avoiding misrecognition of AUG.