A correspondence between solution-state dynamics of an individual protein and the sequence and conformational diversity of its family.

Conformational ensembles are increasingly recognized as a useful representation to describe fundamental relationships between protein structure, dynamics and function. Here we present an ensemble of ubiquitin in solution that is created by sampling conformational space without experimental information using "Backrub" motions inspired by alternative conformations observed in sub-Angstrom resolution crystal structures. Backrub-generated structures are then selected to produce an ensemble that optimizes agreement with nuclear magnetic resonance (NMR) Residual Dipolar Couplings (RDCs). Using this ensemble, we probe two proposed relationships between properties of protein ensembles: (i) a link between native-state dynamics and the conformational heterogeneity observed in crystal structures, and (ii) a relation between dynamics of an individual protein and the conformational variability explored by its natural family. We show that the Backrub motional mechanism can simultaneously explore protein native-state dynamics measured by RDCs, encompass the conformational variability present in ubiquitin complex structures and facilitate sampling of conformational and sequence variability matching those occurring in the ubiquitin protein family. Our results thus support an overall relation between protein dynamics and conformational changes enabling sequence changes in evolution. More practically, the presented method can be applied to improve protein design predictions by accounting for intrinsic native-state dynamics

Metal Binding to Sodium Heparin Monitored by Quadrupolar NMR

Heparins and heparan sulfate polysaccharides are negatively charged glycosaminoglycans and play important roles in cell-to-matrix and cell-to-cell signaling processes. Metal ion binding to heparins alters the conformation of heparins and influences their function. Various experimental techniques have been used to investigate metal ion-heparin interactions, frequently with inconsistent results. Exploiting the quadrupolar 23Na nucleus, we herein develop a 23Na NMR-based competition assay and monitor the binding of divalent Ca2+ and Mg2+ and trivalent Al3+ metal ions to sodium heparin and the consequent release of sodium ions from heparin. The 23Na spin relaxation rates and translational diffusion coefficients are utilized to quantify the metal ion-induced release of sodium ions from heparin. In the case of the Al3+ ion, the complementary approach of 27Al quadrupolar NMR is employed as a direct probe of ion binding to heparin. Our NMR results demonstrate at least two metal ion-binding sites with different affinities on heparin, potentially undergoing dynamic exchange. For the site with lower metal ion binding affinity, the order of Ca2+ > Mg2+ > Al3+ is obtained, in which even the weakly binding Al3+ ion is capable of displacing sodium ions from heparin. Overall, the multinuclear quadrupolar NMR approach employed here can monitor and quantify metal ion binding to heparin and capture different modes of metal ion-heparin binding

Atomic resolution dynamics of cohesive interactions in phase-separated Nup98 FG domains

Cohesive FG domains assemble into a condensed phase forming the selective permeability barrier of nuclear pore complexes. Nanoscopic insight into fundamental cohesive interactions has long been hampered by the sequence heterogeneity of native FG domains. We overcome this challenge by utilizing an engineered perfectly repetitive sequence and a combination of solution and magic angle spinning NMR spectroscopy. We map the dynamics of cohesive interactions in both phase-separated and soluble states at atomic resolution using TROSY for rotational correlation time (TRACT) measurements. We find that FG repeats exhibit nanosecond-range rotational correlation times and remain disordered in both states, although FRAP measurements show slow translation of phase-separated FG domains. NOESY measurements enable the direct detection of contacts involved in cohesive interactions. Finally, increasing salt concentration and temperature enhance phase separation and decrease local mobility of FG repeats. This lower critical solution temperature (LCST) behaviour indicates that cohesive interactions are driven by entropy

Transmembrane signaling and cytoplasmic signal conversion by dimeric transmembrane helix 2 and a linker domain of the DcuS sensor kinase

Transmembrane (TM) signaling is a key process of membrane-bound sensor kinases. The C4-dicarboxylate (fumarate) responsive sensor kinase DcuS of Escherichia coli is anchored by TM helices TM1 and TM2 in the membrane. Signal transmission across the membrane relies on the piston-type movement of the periplasmic part of TM2. To define the role of TM2 in TM signaling, we use oxidative Cys cross-linking to demonstrate that TM2 extends over the full distance of the membrane and forms a stable TM homodimer in both the inactive and fumarate-activated state of DcuS. An S186xxxGxxxG194 motif is required for the stability and function of the TM2 homodimer. The TM2 helix further extends on the periplasmic side into the α6-helix of the sensory PASP domain and on the cytoplasmic side into the α1-helix of PASC. PASC has to transmit the signal to the C-terminal kinase domain. A helical linker on the cytoplasmic side connecting TM2 with PASC contains an LxxxLxxxL sequence. The dimeric state of the linker was relieved during fumarate activation of DcuS, indicating structural rearrangements in the linker. Thus, DcuS contains a long α-helical structure reaching from the sensory PASP (α6) domain across the membrane to α1(PASC). Taken together, the results suggest piston-type TM signaling by the TM2 homodimer from PASP across the full TM region, whereas the fumarate-destabilized linker dimer converts the signal on the cytoplasmic side for PASC and kinase regulation

Depopulation of dense α-synuclein aggregates is associated with rescue of dopamine neuron dysfunction and death in a new Parkinson's disease model.

Parkinson's disease (PD) is characterized by the presence of α-synuclein aggregates known as Lewy bodies and Lewy neurites, whose formation is linked to disease development. The causal relation between α-synuclein aggregates and PD is not well understood. We generated a new transgenic mouse line (MI2) expressing human, aggregation-prone truncated 1-120 α-synuclein under the control of the tyrosine hydroxylase promoter. MI2 mice exhibit progressive aggregation of α-synuclein in dopaminergic neurons of the substantia nigra pars compacta and their striatal terminals. This is associated with a progressive reduction of striatal dopamine release, reduced striatal innervation and significant nigral dopaminergic nerve cell death starting from 6 and 12 months of age, respectively. In the MI2 mice, alterations in gait impairment can be detected by the DigiGait test from 9 months of age, while gross motor deficit was detected by rotarod test at 20 months of age when 50% of dopaminergic neurons in the substantia nigra pars compacta are lost. These changes were associated with an increase in the number and density of 20-500 nm α-synuclein species as shown by dSTORM. Treatment with the oligomer modulator anle138b, from 9 to 12 months of age, restored striatal dopamine release, prevented dopaminergic cell death and gait impairment. These effects were associated with a reduction of the inner density of large α-synuclein aggregates and an increase in dispersed small α-synuclein species as revealed by dSTORM. The MI2 mouse model recapitulates the progressive dopaminergic deficit observed in PD, showing that early synaptic dysfunction is associated to fine behavioral motor alterations, precedes dopaminergic axonal loss and neuronal death that become associated with a more consistent motor deficit upon reaching a certain threshold. Our data also provide new mechanistic insight for the effect of anle138b's function in vivo supporting that targeting α-synuclein aggregation is a promising therapeutic approach for PD

Structural characterization of copper(II) binding to α-Synuclein: Insights into the bioinorganic chemistry of Parkinson's disease

The aggregation of α -synuclein (AS) is characteristic of Parkinson’s disease and other neurodegenerative synucleinopathies. We demonstrate here that Cu(II) ions are effective in accelerating AS aggregation at physiologically relevant concentrations without altering the resultant fibrillar structures. By using numerous spectroscopic techniques (absorption, CD, EPR, and NMR), we have located the primary binding for Cu(II) to a specific site in the N terminus, involving His-50 as the anchoring residue and other nitrogen oxygen donor atoms in a square planar or distorted tetragonal geometry. The carboxylate-rich C terminus, originally thought to drive copper binding, is able to coordinate a second Cu(II) equivalent, albeit with a 300-fold reduced affinity. The NMR analysis of AS–Cu(II) complexes reveals the existence of conformational restrictions in the native state of the protein. The metallobiology of Cu(II) in Parkinson’s disease is discussed by a comparative analysis with other Cu(II)-binding proteins involved in neurodegenerative disorders