The Peripheral Binding of 14-3-3γ to Membranes Involves Isoform-Specific Histidine Residues

Mammalian 14-3-3 protein scaffolds include seven conserved isoforms that bind numerous phosphorylated protein partners and regulate many cellular processes. Some 14-3-3-isoforms, notably γ, have elevated affinity for membranes, which might contribute to modulate the subcellular localization of the partners and substantiate the importance of investigating molecular mechanisms of membrane interaction. By applying surface plasmon resonance we here show that the binding to phospholipid bilayers is stimulated when 14-3-3γ is complexed with its partner, a peptide corresponding to the Ser19-phosphorylated N-terminal region of tyrosine hydroxylase. Moreover, membrane interaction is dependent on salts of kosmotropic ions, which also stabilize 14-3-3γ. Electrostatic analysis of available crystal structures of γ and of the non-membrane-binding ζ-isoform, complemented with molecular dynamics simulations, indicate that the electrostatic potential distribution of phosphopeptide-bound 14-3-3γ is optimal for interaction with the membrane through amphipathic helices at the N-terminal dimerization region. In addition, His158, and especially His195, both specific to 14-3-3γ and located at the convex lateral side, appeared to be pivotal for the ligand induced membrane interaction, as corroborated by site-directed mutagenesis. The participation of these histidine residues might be associated to their increased protonation upon membrane binding. Overall, these results reveal membrane-targeting motifs and give insights on mechanisms that furnish the 14-3-3γ scaffold with the capacity for tuned shuffling from soluble to membrane-bound states.This work was supported by grants from the Norwegian Cancer Society (to ØH), Junta de Andalucía, grant CVI-02483 (to JMSR), The Research Council of Norway (grant 185181 to A.M.), the Western Norway Health Authorities (grant 911618 to A.M.) and The Kristian Gerhard Jebsen Foundation (to AM)

The role of population PK-PD modelling in paediatric clinical research

Children differ from adults in their response to drugs. While this may be the result of changes in dose exposure (pharmacokinetics [PK]) and/or exposure response (pharmacodynamics [PD]) relationships, the magnitude of these changes may not be solely reflected by differences in body weight. As a consequence, dosing recommendations empirically derived from adults dosing regimens using linear extrapolations based on body weight, can result in therapeutic failure, occurrence of adverse effect or even fatalities. In order to define rational, patient-tailored dosing schemes, population PK-PD studies in children are needed. For the analysis of the data, population modelling using non-linear mixed effect modelling is the preferred tool since this approach allows for the analysis of sparse and unbalanced datasets. Additionally, it permits the exploration of the influence of different covariates such as body weight and age to explain the variability in drug response. Finally, using this approach, these PK-PD studies can be designed in the most efficient manner in order to obtain the maximum information on the PK-PD parameters with the highest precision. Once a population PK-PD model is developed, internal and external validations should be performed. If the model performs well in these validation procedures, model simulations can be used to define a dosing regimen, which in turn needs to be tested and challenged in a prospective clinical trial. This methodology will improve the efficacy/safety balance of dosing guidelines, which will be of benefit to the individual child

Characterization of porphobilinogen deaminase mutants reveals that arginine-173 is crucial for polypyrrole elongation mechanism

Abstract Porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthesis, catalyzes the sequential coupling of four porphobilinogen (PBG) molecules into a heme precursor. Mutations in PBGD are associated with acute intermittent porphyria (AIP), a rare metabolic disorder. We used Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to demonstrate that wild-type PBGD and AIP-associated mutant R167W both existed as holoenzymes (Eholo) covalently attached to the dipyrromethane cofactor, and three intermediate complexes, ES, ES₂, and ES₃, where S represents PBG. In contrast, only ES₂ was detected in AIP-associated mutant R173W, indicating that the formation of ES₃ is inhibited. The R173W crystal structure in the ES₂-state revealed major rearrangements of the loops around the active site, compared to wild-type PBGD in the Eholo-state. These results contribute to elucidating the structural pathogenesis of two common AIP-associated mutations and reveal the important structural role of Arg173 in the polypyrrole elongation mechanism

High-affinity anti-Arc nanobodies provide tools for structural and functional studies

Abstract Activity-regulated cytoskeleton-associated protein (Arc) is a multidomain protein of retroviral origin with a vital role in the regulation of synaptic plasticity and memory formation in mammals. However, the mechanistic and structural basis of Arc function is poorly understood. Arc has an N-terminal domain (NTD) involved in membrane binding and a C-terminal domain (CTD) that binds postsynaptic protein ligands. In addition, the NTD and CTD both function in Arc oligomerisation, including assembly of retrovirus-like capsids involved in intercellular signalling. To obtain new tools for studies on Arc structure and function, we produced and characterised six high-affinity anti-Arc nanobodies (Nb). The CTD of rat and human Arc were both crystallised in ternary complexes with two Nbs. One Nb bound deep into the stargazin-binding pocket of Arc CTD and suggested competitive binding with Arc ligand peptides. The crystallisation of the human Arc CTD in two different conformations, accompanied by SAXS data and molecular dynamics simulations, paints a dynamic picture of the mammalian Arc CTD. The collapsed conformation closely resembles Drosophila Arc in capsids, suggesting that we have trapped a capsid-like conformation of the human Arc CTD. Our data obtained with the help of anti-Arc Nbs suggest that structural dynamics of the CTD and dimerisation of the NTD may promote the formation of capsids. Taken together, the recombinant high-affinity anti-Arc Nbs are versatile tools that can be further developed for studying mammalian Arc structure and function, as well as mechanisms of Arc capsid formation, both in vitro and in vivo. For example, the Nbs could serve as a genetically encoded tools for inhibition of endogenous Arc interactions in the study of neuronal function and plasticity

Arc self-association and formation of virus-like capsids are mediated by an N-terminal helical coil motif

Abstract Activity-regulated cytoskeleton-associated protein (Arc) is a protein interaction hub with diverse roles in intracellular neuronal signaling, and important functions in neuronal synaptic plasticity, memory, and postnatal cortical development. Arc has homology to retroviral Gag protein and is capable of self-assembly into virus-like capsids implicated in the intercellular transfer of RNA. However, the molecular basis of Arc self-association and capsid formation is largely unknown. Here, we identified a 28-amino-acid stretch in the mammalian Arc N-terminal (NT) domain that is necessary and sufficient for self-association. Within this region, we identified a 7-residue oligomerization motif, critical for the formation of virus-like capsids. Purified wild-type Arc formed capsids as shown by transmission and cryo-electron microscopy, whereas mutant Arc with disruption of the oligomerization motif formed homogenous dimers. An atomic-resolution crystal structure of the oligomerization region peptide demonstrated an antiparallel coiled-coil interface, strongly supporting NT-NT domain interactions in Arc oligomerization. The NT coil–coil interaction was also validated in live neurons using fluorescence lifetime FRET imaging, and mutation of the oligomerization motif disrupted Arc-facilitated endocytosis. Furthermore, using single-molecule photobleaching, we show that Arc mRNA greatly enhances higher-order oligomerization in a manner dependent on the oligomerization motif. In conclusion, a helical coil in the Arc NT domain supports self-association above the dimer stage, mRNA-induced oligomerization, and formation of virus-like capsids