Malfunction of hERG potassium channels, due to inherited mutations or inhibition by drugs, can cause long QT syndrome, which can lead to life-threatening arrhythmias. A three-dimensional structure of hERG is a prerequisite to understand the molecular basis of hERG malfunction. To achieve a consensus model, we carried out an extensive analysis of hERG models based on various alignments of helix S5. We analyzed seven models using a combination of conventional geometry/packing/normality validation methods as well as molecular dynamics simulations and molecular docking. A synthetic test set with the X-ray crystal structure of K(v)1.2 with artificially shifted S5 sequences modeled into the structure served as a reference case. We docked the known hERG inhibitors (+)-cisapride, (S)-terfenadine, and MK-499 into the hERG models and simulation snapshots. None of the single analyses unambiguously identified a preferred model, but the combination of all three revealed that there is only one model that fulfils all quality criteria. This model is confirmed by a recent mutation scanning experiment (P. Ju, G. Pages, R. P. Riek, P. C. Chen, A. M. Torres, P. S. Bansal, S. Kuyucak, P. W. Kuchel, J. I. Vandenberg, J. Biol. Chem. 2009, 284, 1000-1008). We expect the modeled structure to be useful as a basis both for computational studies of channel function and kinetics as well as the design of experiments.

A conformational analysis of the valine side chains of ribonuclease T1 (RNase T1) was performed using NMR spectroscopy, in particular homonuclear (1H, 1H and 13C, 13C) and heteronuclear (1H, 15N and 1H, 13C) vicinal spin-spin coupling constants as obtained from E.COSY-type NMR experiments. The coupling constants related to the chi 1 dihedral angle in valine, 3JH alpha H beta, 3JNH beta, 3JC'H beta, 3JH alpha C gamma 1, 3JH alpha C gamma 2, 3JC'C gamma 1, and 3JC'C gamma 2, were evaluated in a quantitative manner. The analysis of 3J data allowed for the stereospecific assignment of the valine methyl resonances. On the basis of various models for motional averaging of coupling constants, a fit of the torsion angles chi 1 to a set of the experimental 3J coupling constants (3JH alpha H beta, 3JNH beta, 3JC'H beta) was carried out. The resulting side-chain conformations were examined with respect to NOE distance informations. Single rotameric states emerged for Val16, Val67, Val79, and Val101, while conformational equilibria between staggered rotamers were found for Val33 and Val78. Using a different model approach, Val52 and Val89 are also likely to exhibit unimodal chi 1 angle distributions. The analysis was found to depend critically on the set of Karplus parameters used. Except for Val52 and Val78, the predominant rotamers obtained from 3J coupling informations agree with the conformation in the crystal structure of ribonuclease T1 (Martinez-Oyanedel et al., 1991).

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Recently, a solid-state NMR study revealed that scorpion toxin binding leads to conformational changes in the selectivity filter of potassium channels. The exact nature of the conformational changes, however, remained elusive. We carried out all-atom molecular dynamics simulations that enabled us to cover the complete pathway of toxin approach and binding, and we validated our simulation results by using solid-state NMR data and electrophysiological measurements. Our structural model revealed a mechanism of cooperative toxin-induced conformational changes that accounts both for the signal changes observed in solid-state NMR and for the tight interaction between KcsA-Kv1.3 and Kaliotoxin. We show that this mechanism is structurally and functionally closely related to recovery from C-type inactivation. Furthermore, our simulations indicate heterogeneity in the binding modes of Kaliotoxin, which might serve to enhance its affinity for KcsA-Kv1.3 further by entropic stabilization.

To gain insight into intramolecular carbohydrate-protein interactions at the molecular level, the solution structure of differently deglycosylated variants of the R-subunit of human chorionic gonadotropin have been studied by NMR spectroscopy. Significant differences in chemical shifts and NOE intensities were observed for amino acid residues close to the carbohydrate chain at Asn78 upon deglycosylation beyond Asn78-bound GlcNAc. As no straightforward strategy is available for the calculation of the NMR structure of intact glycoproteins, a suitable computational protocol had to be developed. To this end, the X-PLOR carbohydrate force field designed for structure refinement was extended and modified. Furthermore, a computational strategy was devised to facilitate successful protein folding in the presence of extended glycans during the simulation. The values for φ and ψ dihedral angles of the glycosidic linkages of the oligosaccharide core fragments GlcNAc2( 1-4)GlcNAc1 and Man3( 1-4)GlcNAc2 are restricted to a limited range of the broad conformational energy minima accessible for free glycans. This demonstrates that the protein core affects the dynamic behavior of the glycan at Asn78 by steric hindrance. Reciprocally, the NMR structures indicate that the glycan at Asn78 affects the stability of the protein core. The backbone angular order parameters and displacement data of the generated conformers display especially for the -turn 20-23 a decreased structural order upon splitting off the glycan beyond the Asn78-bound GlcNAc. In particular, the Asn-bound GlcNAc shields the protein surface from the hydrophilic environment through interaction with predominantly hydrophobic amino acid residues located in both twisted -hairpins consisting of residues 10-28 and 59-84.Human chorionic gonadotropin (hCG) 1 is a glycoprotein hormone involved in the maintenance of the corpus luteum during early pregnancy. It is a heterodimer consisting of noncovalently associated R-and -subunits. Both subunits are glycosylated and contain a cystine knot motif formed by three disulfide bridges as a central structural element (1,2). Besides the role of R-subunit (RhCG or RhCG[glycan 52,78 ]) in the R -dimer, it has been discovered that the noncombined R-subunit of hCG (hCG-R f ), which is produced in significant amounts by the placenta during pregnancy, has an independent biological activity in being able to stimulate prolactin secretion from human decidual cells (3, 4).Glycosylation of proteins has been shown to have many functions, including stabilization of the protein structure (5). In the R-subunit of hCG, the two glycosylation sites at Asn52 and Asn78, respectively, have remarkably different properties with respect to the stability of the subunit as shown by sitedirected mutagenesis (6). The absence of glycosylation at Asn52 does not affect folding and stability of the R-subunit. In contrast, mutant R-subunits lacking glycosylation at Asn78 are poorly secreted and rapidly degraded, although the precise molecular origin of thi...

The three-dimensional solution structure of the a-subunit in the a,b heterodimeric human chorionic gonadotropin (hCG), deglycosylated with endo-b-N-acetylglucosaminidase-B (dg-ahCG), was determined using 2D homonuclear and 2D heteronuclear 1 H, 13 C NMR spectroscopy at natural abundance in conjunction with the program package xplor. The distance geometry/simulated annealing protocol was modified to allow for the efficient modelling of the cystine knot motif present in ahCG. The protein structure was modelled with 620 interproton distance restraints and the GlcNAc residue linked to Asn78 was modelled with 30 protein-carbohydrate and 3 intraresidual NOEs. The solution structure of dg-ahCG is represented by an ensemble of 27 structures. In comparison to the crystal structure of the dimer, the solution structure of free dg-ahCG exhibits: (a) an increased structural disorder (residues 33±57); (b) a different backbone conformation near Val76 and Glu77; and (c) a larger flexibility. These differences are caused by the absence of the interactions with the b-subunit. Consequently, in free dg-ahCG, compared to the intact dimer, the two hairpin loops 20±23 and 70±74 are arranged differently with respect to each other. The b-GlcNAc (78) is tightly associated with the hydrophobic protein-core in between the b-hairpins. This conclusion is based on the NOEs from the axial H1, H3, H5 atoms and the N-acetyl protons of b-GlcNAc(78) to the protein-core. The hydrophobic protein-core between the b-hairpins is thereby shielded from the solvent.

The solution structure of the serine protease PB92 presents a well defined global fold which is rigid with the exception of a restricted number of sites. Among the limited number of residues involved in significant internal mobility are those of two pockets, termed S1 and S4, within the substrate-binding site. The presence of flexibility within the binding site supports the proposed induced fit mechanism of substrate binding.

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