A Conserved Hydrophobic Moiety and Helix–Helix Interactions Drive the Self-Assembly of the Incretin Analog Exendin-4

Exendin-4 is a pharmaceutical peptide used in the control of insulin secretion. Structural information on exendin-4 and related peptides especially on the level of quaternary structure is scarce. We present the first published association equilibria of exendin-4 directly measured by static and dynamic light scattering. We show that exendin-4 oligomerization is pH dependent and that these oligomers are of low compactness. We relate our experimental results to a structural hypothesis to describe molecular details of exendin-4 oligomers. Discussion of the validity of this hypothesis is based on NMR, circular dichroism and fluorescence spectroscopy, and light scattering data on exendin-4 and a set of exendin-4 derived peptides. The essential forces driving oligomerization of exendin-4 are helix–helix interactions and interactions of a conserved hydrophobic moiety. Our structural hypothesis suggests that key interactions of exendin-4 monomers in the experimentally supported trimer take place between a defined helical segment and a hydrophobic triangle constituted by the Phe22 residues of the three monomeric subunits. Our data rationalize that Val19 might function as an anchor in the N-terminus of the interacting helix-region and that Trp25 is partially shielded in the oligomer by C-terminal amino acids of the same monomer. Our structural hypothesis suggests that the Trp25 residues do not interact with each other, but with C-terminal Pro residues of their own monomers.

Synthesizing Mechanical Chains for Morphing Between Spatial Curves

This paper extends the kinematic synthesis methodology for designing a chain of bodies to match a set of arbitrary curves to the spatial case. The methodology initiates with an arbitrary set of spatial curves, and concludes with a set of bodies defined by their spatial features. The bodies synthesized can be one of three types: a rigid segment, a helical segment with constant curvature and torsion but varying length, and a growth segment that maintains its geometry but may be scaled to become larger or smaller. To realize mechanical chains for mechanisms that achieve spatial shape change, only rigid and helical segments are used. After designing the segments, they may be aligned with the original spatial curves with their ends connected via an optimization. For two curves, these connections may be made with revolute joints to obtain high accuracy. For three or more curves, spherical joint connections allow for best accuracy. To compare curves as is useful in morphometry, all three segment types may be employed. In this case, an accurate description of the changes between curves is important, and optimizing to connect the segments is not needed. The procedure for redefining the curves in a way that the techniques in this paper may be applied, as well as the methodologies for synthesizing the three segment types are presented. Examples include a continuum robot problem and the morphometric analyses of cochlear curves and the lambdoidal suture located on a human skull. This work extends the established planar techniques for synthesizing mechanisms and addressing morphometric issues that are motivated with curves in two-dimensions.

Synthesizing Mechanical Chains for Morphing Between Spatial Curves

This work investigates the kinematic synthesis methodology for designing a chain of three-dimensional bodies to match a set of arbitrary spatial curves. The bodies synthesized can be one of three types: a rigid segment, a helical segment with constant curvature and torsion but varying length, and a growth segment that maintains its geometry but may be scaled to become larger or smaller. To realize mechanical chains, only rigid and helical segments are used. After designing the segments, they may be aligned with the original spatial curves with their ends connected via an optimization. For two curves, these connections may be made with revolute joints to obtain high accuracy. For three or more curves, spherical joint connections allow for the best accuracy. To compare curves as is useful in morphometry, all three segment types may be employed. In this case, an accurate description of the changes between curves is important, and optimizing to connect the segments is not needed. The procedure for redefining the curves in a way that the techniques in this paper may be applied, as well as the methodologies for synthesizing the three segment types are presented. Examples include a continuum robot problem and the morphometric analyses of chochlear curves and the lambdoidal suture. This work extends the established planar techniques for synthesizing mechanisms and addressing morphometric issues that are motivated with curves in two-dimensions.

Molecular dynamics simulations disclose early stages of the photo-activation of cryptochrome 4

Birds appear to be equipped with a light-dependent, radical-pair-based magnetic compass that relies on truly quantum processes. While the identity of the sensory protein has remained speculative, cryptochrome 4 has recently been identified as the most auspicious candidate. Here, we report on allatom molecular dynamics (MD) simulations addressing the structural reorganisations that accompany the photoreduction of the flavin cofactor in a model of the European robin cryptochrome 4 (ErCry4). Extensive MD simulations reveal that the photo-activation of ErCry4 induces large-scale conformational changes on short (hundreds of nanoseconds) timescales. Specifically, the photo-reduction is accompanied with the release of the C-terminal tail, structural rearrangements in the vicinity of the FAD-binding site, and the noteworthy formation of an α-helical segment at the N-terminal part. Some of these rearrangements appear to expose potential phosphorylation sites. We describe the conformational dynamics of the protein using a graph-based approach that is informed by the adjacency of residues and the correlation of their local motions. This approach reveals densely coupled reorganisation communities, which facilitate an efficient signal transduction due to a high density of hubs. These communities are interconnected by a small number of highly important residues characterized by high betweenness centrality. The network approach clearly identifies the sites restructuring upon photoactivation, which appear as protrusions or delicate bridges in the reorganisation network. We also find that, unlike in the homologous cryptochrome from D. melanogaster, the release of the C-terminal domain does not appear to be correlated with the transposition of a histidine residue close to the FAD cofactor.

A helical segment makes potassium channels go-go

More than 500 variants in the KCNH2 gene, which encodes the cardiac human ether-a-go-go (hERG) ion channel, have been associated with sudden cardiac death, but only a subset of these variants have been investigated. Matthew D. Perry and colleagues now combine NMR spectroscopy and electrophysiological experiments to explore the functional properties of mutations within an overlooked hERG helix, finding important contributions to channel function.

Structure and stability of recombinant bovine odorant-binding protein: III. Peculiarities of the wild type bOBP unfolding in crowded milieu

Contrarily to the majority of the members of the lipocalin family, which are stable monomers with the specific OBP fold (a β-barrel consisting of a 8-stranded anti-parallel β-sheet followed by a short α-helical segment, a ninth β-strand, and a disordered C-terminal tail) and a conserved disulfide bond, bovine odorant-binding protein (bOBP) does not have such a disulfide bond and forms a domain-swapped dimer that involves crossing the α-helical region from each monomer over the β-barrel of the other monomer. Furthermore, although natural bOBP isolated from bovine tissues exists as a stable domain-swapped dimer, recombinant bOBP has decreased dimerization potential and therefore exists as a mixture of monomeric and dimeric variants. In this article, we investigated the effect model crowding agents of similar chemical nature but different molecular mass on conformational stability of the recombinant bOBP. These experiments were conducted in order shed light on the potential influence of model crowded environment on the unfolding-refolding equilibrium. To this end, we looked at the influence of PEG-600, PEG-4000, and PEG-12000 in concentrations of 80, 150, and 300 mg/mL on the equilibrium unfolding and refolding transitions induced in the recombinant bOBP by guanidine hydrochloride.

Structure and stability of recombinant bovine odorant-binding protein: III. Peculiarities of the wild type bOBP unfolding in crowded milieu

Contrarily to the majority of the members of the lipocalin family, which are stable monomers with the specific OBP fold (a β-barrel consisting of a 8-stranded anti-parallel β-sheet followed by a short α-helical segment, a ninth β-strand, and a disordered C-terminal tail) and a conserved disulfide bond, bovine odorant-binding protein (bOBP) does not have such a disulfide bond and forms a domain-swapped dimer that involves crossing the α-helical region from each monomer over the β-barrel of the other monomer. Furthermore, although natural bOBP isolated from bovine tissues exists as a stable domain-swapped dimer, recombinant bOBP has decreased dimerization potential and therefore exists as a mixture of monomeric and dimeric variants. In this article, we investigated the effect model crowding agents of similar chemical nature but different molecular mass on conformational stability of the recombinant bOBP. These experiments were conducted in order shed light on the potential influence of model crowded environment on the unfolding-refolding equilibrium. To this end, we looked at the influence of PEG-600, PEG-4000, and PEG-12000 in concentrations of 80, 150, and 300 mg/mL on the equilibrium unfolding and refolding transitions induced in the recombinant bOBP by guanidine hydrochloride.

Divergent evolution of an atypical S-adenosyl-l-methionine–dependent monooxygenase involved in anthracycline biosynthesis

Bacterial secondary metabolic pathways are responsible for the biosynthesis of thousands of bioactive natural products. Many enzymes residing in these pathways have evolved to catalyze unusual chemical transformations, which is facilitated by an evolutionary pressure promoting chemical diversity. Such divergent enzyme evolution has been observed in S-adenosyl-l-methionine (SAM)-dependent methyltransferases involved in the biosynthesis of anthracycline anticancer antibiotics; whereas DnrK from the daunorubicin pathway is a canonical 4-O-methyltransferase, the closely related RdmB (52% sequence identity) from the rhodomycin pathways is an atypical 10-hydroxylase that requires SAM, a thiol reducing agent, and molecular oxygen for activity. Here, we have used extensive chimeragenesis to gain insight into the functional differentiation of RdmB and show that insertion of a single serine residue to DnrK is sufficient for introduction of the monooxygenation activity. The crystal structure of DnrK-Ser in complex with aclacinomycin T and S-adenosyl-l-homocysteine refined to 1.9-Å resolution revealed that the inserted serine S297 resides in an α-helical segment adjacent to the substrate, but in a manner where the side chain points away from the active site. Further experimental work indicated that the shift in activity is mediated by rotation of a preceding phenylalanine F296 toward the active site, which blocks a channel to the surface of the protein that is present in native DnrK. The channel is also closed in RdmB and may be important for monooxygenation in a solvent-free environment. Finally, we postulate that the hydroxylation ability of RdmB originates from a previously undetected 10-decarboxylation activity of DnrK.

Aromatic–aromatic interactions between residues in KCa3.1 pore helix and S5 transmembrane segment control the channel gating process

The Ca2+-activated potassium channel KCa3.1 is emerging as a therapeutic target for a large variety of health disorders. One distinguishing feature of KCa3.1 is that the channel open probability at saturating Ca2+ concentrations (Pomax) is low, typically 0.1–0.2 for KCa3.1 wild type. This observation argues for the binding of Ca2+ to the calmodulin (CaM)–KCa3.1 complex, promoting the formation of a preopen closed-state configuration leading to channel opening. We have previously shown that the KCa3.1 active gate is most likely located at the level of the selectivity filter. As Ca2+-dependent gating of KCa3.1 originates from the binding of Ca2+ to CaM in the C terminus, the hypothesis of a gate located at the level of the selectivity filter requires that the conformational change initiated in the C terminus be transmitted to the S5 and S6 transmembrane helices, with a resulting effect on the channel pore helix directly connected to the selectivity filter. A study was thus undertaken to determine to what extent the interactions between the channel pore helix with the S5 and S6 transmembrane segments contribute to KCa3.1 gating. Molecular dynamics simulations first revealed that the largest contact area between the pore helix and the S5 plus S6 transmembrane helices involves residue F248 at the C-terminal end of the pore helix. Unitary current recordings next confirmed that modulating aromatic–aromatic interactions between F248 and W216 of the S5 transmembrane helical segment and/or perturbing the interactions between F248 and residues in S6 surrounding the glycine hinge G274 cause important changes in Pomax. This work thus provides the first evidence for a key contribution of the pore helix in setting Pomax by stabilizing the channel closed configuration through aromatic–aromatic interactions involving F248 of the pore helix. We propose that the interface pore helix/S5 constitutes a promising site for designing KCa3.1 potentiators.